The effects of environmental
mycobacteria on VLP and
MVA based vaccines against
tuberculosis
Rahiman Sharief Faiyaz
A thesis submitted for the degree of
Master of Science (Immunology) At the University
of Otago, Dunedin, New Zealand
May 2009
i
ABSTRACT
BCG is already established as a vaccine against a global epidemic of
tuberculosis, but its efficacy remains variable. It has shown almost no protection
against TB in tropical countries like Africa and India. One of the prime reasons
postulated for the failure of BCG as a vaccine is associated with pre-exposure to
environmental mycobacteria. Cross-sensitisation to shared mycobacterial
antigens is regarded as an important factor for this variation in efficacy.
This study investigated the effects of environmental Mycobacterium avium
exposure on immune responses to BCG and two novel TB vaccine candidates :
Rabbit haemorrhagic disease (RHDV) virus-like particles conjugated with
Antigen 85A (VLP/Ag85A) and Modified vaccinia virus Ankara expressing
Antigen 85A (MVA/Ag85A). Ag85A peptide was used because it is known to
be an effective immunogen. M. avium strain WAg206 was chosen for this study
as it has previously been shown to interfere with BCG vaccination. RHDV VLP
were generated using a recombinant baculovirus containing the VP60 capsid
gene. VLP/Ag85A was prepared by chemical conjugation of mycobacterial
peptide Ag85A to VLP. MVA/Ag85A is a genetically modified vaccinia virus
expressing Ag85A. T cell proliferation assays, cytokine assays, total antibody
and antibody isotype assays were carried out after vaccination to measure the
immunogenicity.
The results suggest that among these novel vaccines, MVA/Ag85A is the best
vaccine candidate following pre-exposure to WAg206 as it generated a stronger
Th1 type of immune response than either BCG or VLP/Ag85A based on
proliferation assays and cytokine assays specific to mycobacterial antigens.
High levels of antigen-specific IFN-γ, a Th1 cytokine, were recorded when mice
were vaccinated with MVA/Ag85A following pre-exposure to WAg 206. On
vaccination with VLP/Ag85A, antigen-specific IFN-γ responses were low, but
ii
the presence of higher levels of total antibody specific to mycobacterial antigens
suggested induction of a predominant Th2 response which is not protective.
Future work to improve the cell-mediated response to VLP/Ag85A may include
using an adjuvant which enhances Th1 responses and overcomes the inhibitory
effect of environmental mycobacteria.
iii
ACKNOWLEDGEMENTS
I would like to thank almighty first who has blessed me with this wonderful year and
professional opportunity.
It will be less no matter how much I thank my family for supporting me in every way
possible. Thank you mom for being with me in the times of emotional distress!
It will be an understatement to thank my fantastic supervisors Assoc Prof, Margaret Baird
and Dr. Sarah Young. It was amazing to be your student this year, and will always cherish all
those professional moments as they are very special to me being first time in a foreign nation.
You always made me feel very comfortable discussing any issue. This thesis wouldn’t have
taken a proper shape without you. My heart- felt thanks to both of you!
I would like to thank Michelle Wilson, Lynn Slobbe, Vivian Young from the bottom of my
heart for all the assistance, which actually helped me in learning all the practical work from
scratch. Your support and patience will always be remembered.
Thank you very much my ‘402’ buddies! You guys were real fun. I will be always grateful
for those little things like sharing your blocking buffer!
Kaustabh Roy and Tara, thanks for being such good friends from the start. You made the year
quite memorable with all the fun- filled moments like bowling, home- made dinners, beach
drives, etc,. I would like to thank my flat- mates Anirban, Purba, Praveen, Ram, Ganesh and
Gowtham for the care shown by making yummy food and hot Indian masala tea.
I sincerely thank Dept. of Microbiology, University of Otago for giving me this opportunity
and providing with financial support to finish this project swiftly.
My heartfelt thanks to my friends back home Kartiek, Rajesh and Pavan for their moral
support from the beginning.
I would like to dedicate this work to our beloved Assoc. Prof Gle nn Buchan, who encouraged
me to take this project in the beginning. Your presence will be always missed sir.
A big thank you to everyone who supported me in one way or other!
iv
Table of Contents
1
INTRODUCTION
1
1.1
Tube rculosis and Mycobacte ria
2
Tuberculosis
2
Mycobacterium tuberculosis
2
1.2
BCG
3
1.3
Immune responses to mycobacteria
4
Role of T-cell subsets in mycobacterial infections
7
CD4 T cells
7
NKT cells
10
Immune- mediators of tuberculosis
11
1.4
Curre nt Vaccination strategies
14
1.5
Environmental mycobacteria and their influence on BCG vaccine
15
M. avium WAg 206
18
New Vaccination strategies for MTB
19
Rabbit haemorrhagic disease virus as vaccine carriers
19
Antigen 85 complex
20
MVA/AG85A (Modified vaccinia virus Ankara expressing Ag85A)
20
1.7
Objectives of this study
21
2
MATERIALS & METHODS
22
2.1
Animals
23
2.2
Media
23
1.6
2.3.1 VLP/Ag85 preparation
SDS-PAGE (Polyacrylamide gel electrophoresis)
23
25
2.3.2 MVA/AG85A
26
2.4
Vaccination schedule
26
2.5
Splenocyte suspension preparation
28
v
2.6
Splenocyte culture for cytokine data
29
2.7
Splenocyte proliferation assay
29
2.8
IFN-γ/ IL-2/ IL-5 ELISA of splenocyte culture supernatants
30
2.9
Total serum antibody ELISA (PPD-b / Ag85)
31
2.10 Isotyping serum antibody ELISA (PPD-b / Ag85)
32
2.11 Statistical analysis
33
RESULTS
34
3.1 Preliminary experime nts de monstrating that BCG vaccination
induces immune responses to bovine-PPD and Ag85 in mice
35
3.2
3.3
Proliferation responses
35
Cytokine responses
35
Antibody responses
36
Pre-sensitising mice by oral administration of M. avium Wag206 affects the
immune response to BCG in mice
42
Proliferation responses
42
Cytokine responses
42
Antibody responses
43
VLP can be coupled with Ag85 using chemical conjugation technique
49
3.4 Vaccination with VLP/Ag85 generated low antigen-specific IFN-gamma response
only in mice
51
Proliferation responses
51
Cytokine responses
51
3.5 Pre-senstising mice with M. Avium Wag206 has less effect on antigen-specific
immune response to MVA/AG85A than on those to VLP/Ag85A
54
3.6
vi
Proliferation responses
54
Cytokine responses
54
Antibody responses
55
Summary of results
62
4
DISCUSSION
64
4.1
Protective immune response conferred by BCG
65
4.2
Modulation of immune response by BCG following exposure to environmental
mycobacteria
66
4.3 When pre-exposed to environme ntal mycobacte ria, MVA/AG85A proves to be a
better vaccine candidate than VLP/Ag85A
70
4.4
Future directions
75
5
REFERENCES
77
List of Tables
TABLE 1
Table featuring the major attributes of the immune profiles of Th1 and Th2
cells
TABLE 2
10
Table showing vaccination strategy in different mice groups used in the
study.
26
List of Figures
FIGURE 3.1
Comparison of proliferation of splenocytes from BCG vaccinated mice and
naïve (control) mice with different antigens (PPD-b, Ag85A).
FIGURE 3.2
37
IL-2 expression (Cytokine ELISA) produced by cultured splenocytes from
BCG vaccinated mice and naïve (control) mice in response to in vitro
challenge with with antigens PPD-b and Ag85A at different concentrations
38
vii
FIGURE 3.3
Comparison of IFN-gamma expression (Cytokine ELISA) produced by
cultured splenocytes from BCG vaccinated mice in response to challenge
with different antigens PPD-b, Ag85A at different concentrations
FIGURE 3.4
39
Comparison of IL-5 expression produced by cultured splenocytes from BCG
vaccinated mice versus naïve mice in response to challenge with different
antigens PPD-b, Ag85A at different concentrations
FIGURE 3.5
Comparison of Serum antibody levels to PPD-b obtained from BCG
vaccinated mice group and naïve (control) mice.
FIGURE 3.6
40
41
Comparison of proliferation of splenocytes from BCG vaccinated mice,
BCG vaccinated mice pre-sensitised orally with M. avium strain Wag206
and naïve (control) mice in response to PPD-b, PPD-a and Ag85A
FIGURE 3.7
44
Comparison of IL-2 expression produced by cultured splenocytes from BCG
vaccinated mice , BCG vaccinated mice pre-sensitised orally with M. avium
strain Wag206 and naïve (control) mice in response to different antigens
(PPD-b, PPD-a, Ag85A)
FIGURE 3.8
45
Comparison of IFN-gamma expression (cytokine ELISA) produced by
cultured splenocytes from BCG vaccinated mice, BCG vaccinated mice presensitised orally with M. avium strain Wag206 and naïve (control) mice in
response to different antigens (PPD-b, PPD-a, Ag85A)
FIGURE 3.9
46
Comparison of IL-5 production by cultured splenocytes from BCG
vaccinated mice , BCG vaccinated mice pre-sensitised orally with M. avium
viii
strain Wag206 and naïve (control) mice in response to different antigens
(PPD-b, PPD-a, Ag85A)
FIGURE 3.10
47
(a). Comparison of Serum total-antibody levels specific to PPD-b obtained
from BCG vaccinated mice, BCG vaccinated mice pre-sensitised orally with
M. avium strain Wag206 and naïve (control) mice in response to PPD-b and
PPD-a
48
(b). Comparison between levels of PPD-b specific IgG1, IgG2a antibody
response and PPD-a specific IgG1, IgG2a antibodies in sera obtained from
BCG vaccinated mice , BCG vaccinated mice pre-sensitised orally with M.
avium strain Wag206 and naïve (control) mice in response to PPD-b and
PPD-a
48
FIGURE 3.11 Chemically conjugated VLP/Ag85AA run on a 10% SDS-PAGE gel along
with VP60 (VLP only) and titrations of BSA used as positive control
50
FIGURE 3.12 Comparison of proliferation of splenocytes from VLP alone (VP60)
administered mice and VLP/Ag85A vaccinated mice in response to PPD-b,
PPD-a and Ag85A
52
FIGURE 3.13 Comparison of IFN-gamma expression produced by cultured splenocytes
from VP60 (VLP only)-vaccinated mice and VLP/Ag85A-vaccinated mice
in response to antigens PPD-b, PPD-a and Ag85A
53
FIGURE 3.14 (a). Comparison of proliferation of splenocytes (CPM) of different
vaccination groups (BCG, VLP only, VLP/Ag85AA, MVA/AG85A) presensitised with M. avium strain Wag206 and PBS (control mice) in
response to PPD-b, PPD-a and Ag85A
ix
57
FIGURE 3.15 Comparison of IL-2 expression in different vaccination groups (BCG, VLP
only, VLP/Ag85AA, MVA/AG85A) pre-sensitised with M. avium strain
Wag206 and PBS (control mice) in response to PPD-b, PPD-a and Ag85A
58
FIGURE 3.16 Comparison of IFN-gamma expression in different vaccination groups
(BCG, VLP only, VLP/Ag85AA, MVA/AG85A) pre-sensitised with M.
avium strain Wag206 and PBS (control mice) in response to PPD-b, PPD-a
and Ag85A
59
FIGURE 3.17 Comparison of IL-5 expression in different vaccination groups (BCG, VLP
only, VLP/Ag85AA, MVA/AG85A) pre-sensitised with M. avium strain
Wag206 and PBS (control mice) in response to PPD-b, PPD-a and Ag85A
60
FIGURE 3.18 (a). Comparison of total serum antibody levels specific to PPD-b and
Ag85A through 1 in 20 dilution of sera obtained from in different
vaccination groups (BCG, VLP only, VLP/Ag85AA, MVA/AG85A) presensitised with M. avium strain Wag206 and PBS (control mice)
61
(b). Comparison between levels of PPD-b specific IgG1, IgG2a antibodies
and PPD-a specific IgG1, IgG2a antibodies (in ng/mL) in sera obtained
from in different vaccination groups (BCG, VLP only, VLP/Ag85AA,
MVA/AG85A) pre-sensitised with M. avium strain Wag206 and PBS
(control mice).
x
61
List of Abbreviations
Ag85
Antigen 85
AIDS
Acquired Immunodeficiency Syndrome
APC
Antigen presenting cell
BCG
Bacille Calmette-Geurin
CD
Cluster of differentiation
cDMEM
Complete Dulbecco’s Modified Eagle’s Medium
CFU
Colony forming units
CPM
counts per minute
CsCl
Caesium chloride
DC
Dendritic cell
DMF
Dimethyl formamide
DPBS
Dulbecco’s PBS
ELISA
Enzyme Linked Immunosorbant Assay
FCS
Foetal calf serum
HIV
Human Immunodeficiency Virus
HRP
Horse radish peroxidise
IFN-γ
Interferon- gamma
IgG
Immunoglobulin
IL-12
Interleukin-12
IL-2
Interleukin-2
IL-5
Interleukin-5
kDa
kiloDalton
MHC
Major histocompatibility complex
ml
millilitres
MOI
Multiplicity of infection
xi
MTB
Mycobacterium tuberculosis
NK
Natural killer cells
NO
Nitric oxide
NOS
Nitric oxide synthase
PBS
Phosphate buffered saline
PenStrep
Pencillin and Streptomycin
PPD-a
Purified protein derivative – avium
PPD-b
Purified protein derivative - bovine
RHDV
Rabbit haemorrhagic disease virus
RNI
Reactive nitrogen intermediates
SDS-PAGE
Sodium dodecyl sulphate – polyacrylamide gel electrophoresis
SMCC
4-[N-maleimidomethyl]cyclohexane-1-carboxylate
TB
Tuberculosis
Tc
Cytotoxic T lymphocyte
Th1
T helper 1 type cell
Th2
T helper 2 type cell
TNF-α
Tumour necrosis factor – alpha
VLP
Virus like particle
WAg
Wallaceville AgResearch
XDR
Extreme drug resistant
xii
xiii
Chapter-1
INTRODUCTION
1
1.1 TUBERCULOSIS and M YCOBACTERIA
Tube rculosis
Tuberculosis (TB) is a contagious bacterial disease caused by Mycobacterium tuberculosis
(MTB). It is normally a pulmonary disease mainly affecting lungs although it is capable of
infecting other systems of body such as the lymphatic system, central nervous system, and the
circulatory system.
TB is considered as a major cause of death worldwide, and was responsible for more than 1.6
million deaths in the year 2005 (WHO, 2007).
A major cohort of patients that die from TB are HIV-positive. A subject who is HIV-positive
and has TB infection has a higher risk of morbidity when compared with a subject who is
only infected with TB (WHO, 2007).
In addition to the increase in incidence of death from TB with HIV infection there are now
MTB(M. tuberculosis) strains that are resistant to the anti-TB drugs isoniazid and rifampicin,
the two most powerful anti- TB drugs. There is also extreme drug-resistant (XDR) TB, where
no known antibiotic is efficacious (WHO, 2007).
Mycobacterium tuberculosis
MTB is a non-motile rod-shaped bacterium and belongs to the Actinomycetes family. The
rods are 2-5 micrometers in length (Shinnick & Good, 1994). MTB is an obligate aerobe. For
this reason, in the classic case of tuberculosis, MTB complexes are always found in the wellaerated upper lobes of the lungs. The bacterium is a facultative intracellular parasite, usually
of macrophages, and has a slow doubling time, 24 hours, a physiological characteristic that
2
may contribute to its virulence (Cole & Brosch et al., 1998). Chains of bacterial cells in
smears made from in vitro grown colonies often form distinctive serpentine cords. This
observation was first made by Robert Koch who associated cord factor with virulent strains
of the bacterium. MTB is not classified as either Gram-positive or Gram-negative because it
does not have the chemical characteristics of either, although the bacteria do contain
peptidoglycan in their cell wall.
Mycobacterium species are classified as acid- fast bacteria due to their impermeability to
certain dyes and stains. However, acid- fast bacteria will retain dyes when heated and treated
with acidified organic compounds. One acid-fast staining method routinely used for MTB is
the Ziehl-Neelsen stain.
1.2 BCG
BCG (Bacillus-Calmette-Guerin) was first developed by Calmette and Guerin by repeated
sub-culturing of virulent Mycobacterium bovis strain isolated from a bovine mastitis sample
230 times on glycerinated bile potato medium, resulting in gene deletions and duplication and
attenuation. BCG was first administered as a vaccine in 1921 (Oettinger et al., 1999) and it
has become since then the most widely administered vaccine globally.
Later different local sub-strains of BCG evolved (by separate closed activity) such as BCG
Connaught, Danish, Glaxo, Pasteur, and Tokyo, all of which contain differences in terms of
genetic and antigenic composition (Behr, 2001, Oettinger et al., 1999).
To date, more than three billion doses of BCG have been administered worldwide, making it
the most widely employed vaccine in the world. It is a safe vaccine and can be given at birth
(Cassanova et al., 1996). The cheap production cost makes it an ideal, cost-effective
preventive measure against TB as compared to chemotherapy, which is expensive and time
consuming.
3
Most importantly, BCG is the best vaccine tested against tuberculosis to date, as it has been
shown to elicit a strong protective Type 1 immune response (Silva et al., 1999). It is currently
given intra-dermally as a live, attenuated vaccine, as studies have shown that heat-killed
bacteria elicit a different and ineffectual immune response (Hook et al., 1996).
1.3 Immune responses to mycobacteria
Protective immunity against MTB depends on the generation of a Type 1 cellular immune
response, characterized by the secretion of interferon-γ (IFN-γ) from antigen-specific T cells
(McShane et al., 2004). The human immune system relies on both non-specific innate
immunity and antigen-specific acquired immunity working together in concert to protect the
body against these pathogens. The innate system which includes mononuclear phagocytes,
natural killer cells, interferon, and complement, has to be able to recognize pat hogens and
signal the presence of danger to cells of the acquired immune system (Matzinger, 2002;
Janeway & Medzhitov, 2002). The acquired immune system, in turn, is responsible for
further driving the effector immune response to eradicate the MTB. It also needs to form
long- lived immunological memory for enhanced recall responses in order to provide life- long
immunity to this pathogen.
There are two APCs involved in generating a protective immune response to MTB i.e.,
macrophages and dendritic cells (DC).They each have a unique role to play in this response.
DC are the only cells that prime naive T cells, whereas macrophages are the cells that are
primarily infected with MTB and therefore need to kill the bacteria.
4
In MTB infections, alveolar macrophages are the first cell line to encounter bacteria as the
main route of infection is via the respiratory tract (Hingley-Wilson et al., 2003; Nicod et al.,
2000). Macrophages have the microbicidal armoury to destroy most invading pathogens
(reviewed by Raja, 2004). They will phagocytose mycobacteria, contain it with in a
phagosome, and attempt to kill it by fusing the phagosome with a lysosome (Duclos &
Desjardins, 2000; Vieira et al., 2002). Lysosomes contain a variety of toxic substances that
are lethal to bacteria, for example acids, degrading enzymes such as hydrolases and
peroxidases, oxygenated lipids, fatty acids, and reactive oxygen and nitrogen intermediates
(Klebanoff, 2005; Nathan & Shiloh, 2000). However MTB has the ability to resist being
killed in several ways, including inhibition of phago- lysosomal fusion, Reactive oxygen
intermediates (ROI) and Reactive nitrogen intermediates (RNI) production, macrophage
activation and antigen presentation.
Phago- lysosomal function can be constrained in macrophages infected with MTB and there
are different ways to mediate this infection. Phagosomes containing live, viable mycobacteria
are repressed from fusing with lysosomes as fusion of the phagosome with the lysosome has
been shown to be withdrawn (Armstrong & Hart, 1971; Mwandumba et al., 2004). However,
phagosomes containing mycobacteria are capable of fusing with other endosomes, which
suggests they are dynamic, fusion-competent structures that are selectively prevented from
fusing with lysosomal compartments (Russel et al., 1996; Mwandumba et al., 2004). A host
protein found in lymphoid and myeloid cells called tryptophan aspartate-conating coat
(TACO) protein has been identified, and may inhibit fusion. TACO relocalises within cells to
associate with phagosomal membranes containing live MTB and is retained for longer
periods (Ferrari et al., 1999).
MTB are also able to prevent damage caused by ROI and RNI produced by infected
macrophages. Mycobacterial products such as LAMs and Phenoliglycolipid-1 can act as free5
radical scavengers and mitigate oxidative damage (Chan et al., 1989). MTB possesses the
genes noxR1, noxR3 and ahpC that can provide resistance to nitrosative and oxidative stress
(Ehrt et al., 1997; Ruan et al., 1999). Furthermore, MTB expresses a methioinine sulphoxide
formed from the reaction of ONOO- and methionine residues in mycobacterial proteins and
can repair damage caused by RNI (St John et al., 2001).
If the macrophage is properly activated by IFN-γ, it can successfully eliminate or limit the
growth of MTB (Ma et al., 2003). Alternatively, MTB persist in the macrophage due to its
most evolved mechanisms to survive macrophage effector functions (Houben et al., 2006).
Apoptosis, also known as programmed cell death is one more important defence mechanism
against mycobacteria (Keane et al., 2000). Apoptosis prevents the spread of mycobacterial
infection by sequestering the mycobacteria within membrane bound apoptotic bodies
(Fratazzi et al., 1999). The ability to induce apoptosis has been shown to be dependent on the
virulence of the strain involved and perhaps the multiplicity of infection (Danelishville et al.,
2003) (Lee et al., 2006). So it is to be noted that macrophage’s ability to control MTB is
extremely important, as this will influence the outcome of the infection (Houben et al.,
2006)(Flynn and Ernst, 2000).
DC are another type of APC with a characteristic morphology of branched projections called
dendrites, hence their name. Dendritic cells are primarily involved in anti- mycobacterial T
cell immune response, and these are highly represented in sites of MTB infection at the onset
of the inflammatory change (Giacomini et al., 2001).
6
Role of T-cell subsets in mycobacterial infections
The two main subsets of T cells are T helper cells (Th) cells, which characteristically express
the cluster of differentiation 4 (CD4) glycoprotein; and T Cytotoxic (Tc) cells, which express
the CD8 marker on its surface. CD4 and CD8 are both co-receptors for the T cell receptor.
Other T cell subsets include γδ T cells, natural killer T cells, and regulatory T cells (Treg). In
mycobacterial infection, these T cell subsets are known to contribute to protection (Stenger &
Modlin, 1999; Mogues et al., 2001).
CD4 Tcells
The host-pathogen interaction in MTB is usually based on communication between T cells
and infected macrophages. The result of this interplay may lead to control of the infection or
activation of the disease. CD4+ T cells have crucial role in limiting the MTB bacilli growth
with the help of macrophages.
.MTB specific CD4 T-cells are primed in the lymph node by DC expressing MTB antigens
such as Ag85A,B,C, ESAT-6 and CFP-10. These immunodominant antigens are secreted and
hence only induce protective immune responses against live bacteria. When activated by
MTB, CD4+ T cells secrete the macrophage-activating cytokines like IFN-γ and TNF-α
which help macrophages in limiting the growth of intracellular mycobacteria, as those
cytokines are cytotoxic for those infected macrophages.
At present, the most widely accepted theory regarding the immune functioning of Th cells is
the model proposed by Mosmann et al in 1996. According to this model, CD4 T cells can be
subdivided into two independent subsets, Th1 or Th2 cells, o n the basis of cytokine secretion
7
and bioactivities (Mosmann et al, 1986, Coffman, 2006). Each subset has very distinct
effector functions. Th1 cells secrete large amounts of pro- inflammatory cytokines, such as
IL-2, IL-12, IFN-γ, TNF-β and TNF-α upon activation. These cytokines are essential for the
smooth functioning of cell- mediated immune response against intracellular pathogens, such
as viruses and mycobacteria (Liew, 2002). By contrast, Th2 cytokines regulate humoral or
antibody responses by B cells. A Th2 immune response is associated with allergic reactions,
parasitic and extracellular bacterial infections (Murphy et al., 1999; Abbas et al., 1996).
Both Th1 and Th2 cells have a cross-regulatory role, where only one pathway pre-dominates
at any one time (Coffman, 2006). Cytokines of a certain subtype will promote the expansion
of that particular subtype, while inhibiting the development of the other subset. This in turn
induces the development of distinctive effector function specific to the immunogen (Murphy
& Reiner, 2002) e.g. the sequential action of IFN-γ and IL-12 leads to the development of
Th1 cells, while down-regulating Th2 responses. Depending on the balance of Th1 versus
Th2 cytokines produced during immunity, the immune system can be skewed to either a cellmediated pathway or humoral pathway (Coffman, 2006).
It has been well established that CD4 T cells provide protection aginst MTB. Following reinfection by MTB, contact between recirculating T cells and the mycobacteria should ideally
lead to rapid cytokine secretion by memory T cells, at a lower threshold of antigen
stimulation. This will result in the rapid accumulation of moncytes and other immune cells to
the infection site to eliminate pathogen (Reinhardt et al., 2001).
CD8+ T cells (Tc) are another subset of T cells which can be activated in response to MTB
and BCG (Szereday et al, 2002). These cells secrete IFN-γ as well, but in lower quantities
than CD4+ T cells. It is established that CD8+ T cells offer protective immunity by he lping
8
macrophages in controlling intracellular mycobacteria in later stages of infection (Szereday et
al., 2002). Once activated, Tc cells become effector cells that can directly lyse target cells via
cytotoxic granules such as perforin and granzymes (Serb ina et al., 2000), or induce apoptotis
in cells infected intracellularly with mycobacteria (Bonato et al., 1998; Silva et al., 2000). It
was demonstrated by Lazrevic et al (2005) that CD8+ cells in the granuloma periphery
possessed high lytic ability, but produced minute amounts of IFN-γ during acute TB
infection. However in chronic TB infection, this switched to high IFN- γ production, with
low lytic ability (Lazrevic et al., 2005). This shows the dynamic activity of CD8+ cells with
in granuloma.
γδ TCR+ T cells are characterised by unique T cell antigen receptor comprised of γ and δ
chains and mainly express Vδ9 and Vδ2 elements. These cells also have the function of
killing infected macrophages by IFN-γ secretion. Unlike the previously mentioned subsets, γδ
TCR+ T cells (Vδ2+ T cells) recognise different types of mycobacterial molecules based on
phospho antigens which are processed and presented to these cells with co-stimulation by
macrophages (Boom et al., 2003). The prominent role of γδ T cells in protective immunity is
also established by the fact that number and reactivity to phosphate antigens (TUBAg3-4,
isopentyl- ATP, Pyrophosphate molecules such as TUBag1-2, isopentyl pyrophosphate(IPP),
mon-ethyl pyrophosphate(MEPP), and others) increases during primary infection and
challenging after BCG vaccination (Boom et al., 2003).
NKT cells
NKT cells tend to secrete large amounts of both Th1 and Th2 cytokines on stimulus and
establish the ability to regulate immune function. Remarkably, in some disease models, proinflammatory immune responses are modulated by NKT cells (Stock & Akbari, 2008). NKT
9
cells are also known to induce functionally distinct immune responses depending on the
conditions of activation (Wang et al., 2008). The previous studies shows that CD4– NKT
cells from human peripheral blood are Th1-biased, whereas the CD4+ subset produced both
Th1 and Th2 cytokines. Thus, the CD4+ and CD4– NKT-cell subsets may be accountable for
different after effects. It has been established that T and NKT cells play a vital role in
immunity against TB (Apostolou et al., 1999). Increased NKT cell counts indicate the
severity of pulmonary tuberculosis whereas decreased counts imply wrong diagnosis of the
disease (Zahran et al., 2006).
Function
Th1
Th2
Type of immune response
Cell-Mediated
Humoral
Type of cytokines
Pro-inflammatory
Anti-inflammatory
Action
Protection against
Extracellular pathogens
intracellular pathogens
(Bacteria, Protozoa etc)
(MTB, virus, etc)
IFN-γ , TNF-α, IL-2, Il-12
Cytokines
IL-4, IL-5, IL-6, IL-10
Table 1. Table featuring the major attributes of the immune profiles of Th1 and Th2 cells
10
Immune mediators in Tuberculosis
Inte rleukin 2
Interleukin 2 (IL-2) and its receptor (IL-2R) were the first cytokine and cytokine receptor to
be cloned. The first function attributed to IL-2 was a potent capacity to enhance in vitro Tcell proliferation and differentiation. IL-2 was therefore originally named T-cell growth
factor (TCGF). Bachmann & Oxenius (2007) showed that IL-2 also allows for the
differentiation of naive T cells into effector and memory cells.
IL-2 seems to have strong effect on bacterial cell counts after BCG infection in mice. The
decrease in viable bacteria counts indicates that IL-2 affects the replication of organisms not
only the degradation of dead organisms. There might be several pathways for IL-2 to
perform its action. It is reported that IL-2 ceases the replication of MTB in human
macrophages (Jeevan & Asherson, 1988).
IL-2 may also act indirectly through a T cell on macrophages via by inducing the release of
IFN-γ from activated lymphocytes, as it is known that IFN-γ accelerates elimination of
mycobacteria by macrophages. IL-2 may also enhance an effective immune response by
inactivating suppressor cells (Jeevan & Asherson, 1988).
IL-2 can constrain pro-inflammatory IL-17 production and exert its immunosuppressive
function by stimulating the generation and homeostasis of regulatory T cells (T-reg). Indeed,
IL-2 is a non-redundant factor for the in vivo homeostasis of T-reg, which constitute a
fundamental part of immunological self-tolerance and immune regulation . (Bachmann
& Oxenius, 2007).
11
Inte rferon- γ
IFN-γ can be secreted by Th1 cells, Tc cells and NK cells (Emoto et al., 1999, Demangel et
al., 1999). Also known as immune interferon, IFN-γ is the only Type II interferon. It is
serologically distinct from Type I interferons as it is acid- labile, while the Type I variants are
acid-stable. (Schroder et al., 2004)
IFN-γ has shown anti- viral, anti-tumour and immunoregulatory properties (Schroder et al.,
2004). IFN-γ promotes Th1 differentiation by up-regulating the transcription factor T-bet
(Andre A. Lighvani et al, 2001). IFN-γ is the prominent cytokine of Th1 cells.
IFN-γ is a crucial cytokine for the control of mycobacterial infections because IFN-γ plays a
critical role in the stimulation of macrophages to produce nitric oxide (NO), and it is
demonstrated that NO plays a pivotal protective role in MTB infection (Cooper et al, 2002)
because granulomas from NOS2-deficient mice had reduced acid phosphatase activities
suggesting that NO is required for macrophage activation. It is also established that absence
of NOS2 affects the cytokine production of the Th1 type of immune response, ultimately
leading to death (Garcia et al., 2000). It was established to be important for a vaccine to
induce an early response characterized by the production of IFN-γ (Walravens et al., 2002).
IL-12
The importance of interleukin 12 (IL-12) for the generation of protective immune response
during primary infection with mycobacteria has been well estab lished from various murine
and human epidemiological studies (reviewed by Trincheiri, 2003).
IL-12 is the primary cytokine that drives a Type 1 T helper cell immune response in MTB
infections (Dong and Flavell, 2001). It promotes the differentiation of naive T cells into Th1
cells; stimulates the proliferation of T cells; and sustains the Th1 response (Park et al., 2000,
12
O’Shea and Paul, 2002). This allows the formation of a stronger effector T cell response and
long term protection against TB (Stobie et al., 2000). IL-12 also stimulates the growth of
activated CD8 T cells and NK cells, both of which contribute towards protection in TB
infection (Serbina et al., 2001, Xing et al., 2000).
In addition, IL-12 triggers the production of important pro- inflammatory cytokines such as
IFN-γ and TNF-α by activated T cells, APCs and NK cells. IL-12 conveniently suppresses
IL-4 induced IgE production, thus helping to skew the immune response away from a Th2
pathway (Yoshimoto et al., 1997) which is really helpful when the host immune system is
fighting intracellular pathogens such as MTB, where a strong, inflammatory Th1 response is
necessary for protection (Gately et al., 1998).
TNF-α
Tumor Necrosis Factor-alpha (TNF-α) is an important pro-inflammatory cytokine
predominantly released by macrophages, monocytes, dendritic cells, neutrophils, T cells, NK
cells and B cells. TNF-α plays multiple roles in the immune and pathological responses
during mycobacterial infections (Henderson et al., 1997). It works accordingly with IFN-γ to
activate macrophages to inhibit growth of MTB via the induction of NOS2 expression
(Saunders et al., 2005). TNF-α also greatly increases the phagocytic activity and production
of pro- inflammatory cytokines such as interleukin 1 (IL-1) by macrophages (Flynn & Chan,
2001). Sustained autocrine activation of macrophages by this cytokine is not only essential to
enhance the clearance of MTB in acute TB infections, but also to prevent reactivation of
latent disease (Mohan et al., 2001; Scanga et al., 1999).
13
TNF-α also influences T cell responses in a number of ways (Watts, 2005). It plays an
important role in initiating and sustaining T cell responses, and therefore promotes long lived
immunity against MTB (Croft, 2003). Other roles of TNF-α in MTB infections include
inducing T cell colony formation, enhancing cytotoxic T cell and NK cell activity, and
inducing apoptosis in mature T cells.
1.4 Curre nt Vaccination strategies
The only commercially available vaccine for TB is Bacille Calmette Guerin, a live attenuated
strain of M.bovis. Unfortunately, the effectiveness of BCG is inconsistent. BCG vaccination
can effectively prevent forms of childhood TB involving dissemination (Rodrigues et al.,
1993), probably due to its ability to control blood-borne bacilli (Weigeshaus et al., 1989).
However, it is estimated that effectiveness of the BCG vaccine in adults is approximately
50% (Colditz et al., 1994). The effectiveness varies substantially between zero to 80%
protection. Additionally, in some studies (Sterne et al., 1998; Comstock, 1994) the
effectiveness of BCG wanes over time, whereas in others similar levels of protection have
been observed up to 60 years of vaccination (Aronson et al., 2004).
There are several possible explanations for the variation in effectiveness between BCG
vaccinated populations. There are multiple substrains of BCG in use, due to passaging in
different laboratories, which have diverged genetically and immunologically from one
another (Oettinger et al., 1999). Different BCG substrains can confer different levels of
protection against TB (Gheorgiu & Labrange, 1983). Variation may also be a result of host
genetics, with genetic elements prevalent in different populations contributing to the variable
protection afforded by BCG. Moreover, the virulence of different strains of MTB can vary
14
and the virulence of the strain of MTB endemic to a population could affect the effectiveness
of the BCG vaccine.
Finally, there may be environmental factors that affect the effectiveness of the BCG vaccine.
Populations that exhibit low BCG effectiveness are commonly in developing countries, which
have greater exposure to helminth infections and environmental mycobacteria than
populations in developed countries (Elias et al, 2006). Helminth infection at the time of BCG
vaccination is hypothesized to result in either immunosupression of the BCG immune
response or skew the response to an unprotective Th2 profile. Similar hypotheses have been
suggested for the interaction between environmental mycobateria and BCG vaccination
(Rook et al., 2005).
1.5 Environmental mycobacteria and their influence on BCG vaccine
Environmental mycobacteria are omnipresent inhabitants of the environment (Primm et al.,
2004). They can be found in a wide variety of environmental sources such as water, soil, air,
and food sources, and lead life as saprophytes, commensals or symbionts (Yoder et al., 1999).
Environmental mycobacteria are not like other members of MTB complex, as they are not
regarded as obligate pathogens (Herdman & Steele, 2004).
Humans generally tend to expose to these organisms from environmental sources. Water is
considered as the primary source of mycobacterial infection in humans (Vaerewijck et al.,
2005), and this infection happens mainly by aerosols produced during the daily activities such
as drinking, bathing and swimming. Poor hygiene practises are also a common way of getting
infection via food and soil contaminated with environmental mycobacteria (Reed et al, 2006).
15
The Mycobacterium avium complex (MAC) comprises a heterogeneous group of closely
related acid fast organisms that includes M. avium, Mycobacterium intracellulare, M. avium
subspecies paratuberculosis (MAP), M. avium subspecies silvaticum (the 'wood pigeon'
bacillus) and Mycobacterium lepraemurium. Subspecies of M. avium are known to harbour
multiple copies of a number of different genomic insertion elements. Those characterised to
date include IS900 IS901/902, IS1110, IS1245, IS1311 (GenBank accession number
U16276), IS1626 and IS1612. Most of these insertion elements are common to strains of M.
avium that exist ubiquitously in the environment. They rarely cause disease in
immunocompetent hosts (Laochumroonvorapong et al, 1996). However, unique possession of
IS900 or IS901 by M. avium paratuberculosis and certain strains of M. avium respectively,
divides the species into at least two clearly distinct biological subtypes which differ in terms
of their host range and pathogenicity (Kunze et al, 1992). In the absence of any reports
confirming the replication of either M. avium paratuberculosis or IS901+ M. avium in the
environment, it is generally assumed that these organisms exist as obligate pathogens.
Exposure, usually by ingestion, can result in infection of the intestine leading to chronic
inflammatory enteritis in ruminants and various monogastric species including primates.
Given the very close genetic relationship between the different subspecies of M. avium (up to
98% genomic homology), the genetic basis for these biological differences is unclear.
However, the observation that IS901 in M. avium concurs with increased pathogenicity for
experimentally infected mice is supported by several reports of insertion element- mediated
phenotypic change in other bacterial species (Pseudomonas, Streptococcus, Mycobacterium,
Legionella, Escherichia and Yersinia) (Inglis et al., 2006).
There are currently two main hypotheses regarding the effects of cross-senstisation on the
variable efficacy of BCG Vaccination. A famous large scale study involving guinea pigs
16
(Palmer & Long., 1966) suggested that prior exposure to environmental mycobacteria may
lead to natural immunity to other strains of mycobacteria. This may be beneficial to the host
but it also limits the protection that BCG can offer, which may explain its variable efficacy in
different places of the world. However, this degree of natural immunity is obviously
insufficient to protect against virulent MTB infection, as evidenced by high disease burdens
in places where there is also widespread senstisation to environmental mycobacteria, such as
South Africa and India (Black et al., 2002 & 2001).
Another widely acknowledged hypothesis is that prior exposure to environmental
mycobacteria can prime the host’s immune system against shared mycobacterial antigens
(Brandt et al. 2002). This subsequently interferes with the immune response elicited towards
BCG after vaccination, ultimately leading to its failure as a vaccine. This was demonstrated
experimentally in mouse studies by Brandt et al., (2002) which revealed that certain M.avium
strains from Malawi, a region where BCG does not confer protection, affected in vivo BCG
multiplication in mice. Their study revealed that prior exposure to live environmental
mycobacteria can result in a wide range of immune response that is recalled immediately
after BCG vaccination and controls the replication of the vaccine. In their experiments
involving sensitized mice, BCG elicited only a transient immune response with a marginal
frequency of mycobacterium-specific cells and almost no protective immunity against TB. In
contrast, the efficacy of TB subunit vaccines was unaffected by prior exposure to
environmental mycobacteria (Brandt et al., 2002).
Human epidemiological evidence supporting this hypothesis is also available in the form of
several large scale vaccination trials, in which BCG failed to provide any protection to
populations in areas with widespread environmental sensitization (such as Egypt, India and
17
Africa) as compared to places with minimal exposure to environmental mycobacteria (such
as Denmark) (Anderson & Doherthy, 2005).
From these observations, the potential of environmental mycobacteria (in particular the
M.avium species) to interfere with the efficacy of BCG is clearly evident from many human
and livestock vaccination programs, as well as laboratory experimental trials. However, the
immunological mechanisms underlying these findings have yet to be completely elucidated.
A much better understanding of how certain environmental mycobacterial strains affects the
human immune system is thus needed in order to uncover the reasons behind the widespread
failure of BCG in humans. This information will also be extremely valuable for the
development of new or improved vaccines against TB that will be effective in areas of the
world where BCG has failed.
M. avium WAg 206
The study described in this thesis utilises a unique strain of environmental mycobacteria M.
avium strain WAg 206, a IS901 positive strain, isolated from tuberculosis lesions in farmed
cattle by researchers at AgResearch, Wallaceville, New Zealand (de Lisle et al., 2005). WAg
206 affected the performance of BCG to induce a protective immune response in vivo in
different animal models, including cattle, guinea pigs, and mice (Buddle et al., 2002, de Lisle
et al., 2005). Recent studies have shown that following oral infection, M.avium WAg 206
strain had the capacity to invade and persist much longer within the gastrointestinal tract
lymphatic tissues of guinea pigs (de Leslie et al., 2005) and mice (Young et al.,2007) and
induce a strong Th2 immune response.
18
1.6 New Vaccination strategies for MTB
Since the BCG fails to consistently protect against TB, for the reasons described above
significant effort has been directed towards developing new vaccine strategies against TB
testing them in situations where BCG does not work.
Such novel vaccines may include :
Virus like particles coupled with the mycobacterial antigen 85 (VLP-Ag85A) and
recombinant modified vaccinia virus Ankara expressing antigen 85A (MVA85A).
Rabbit haemorrhagic disease virus as vaccine carriers
Virus-like particles represent a specific class of subunit vaccine that mimic the structure of
authentic virus particles. They can be helpful in presenting antigenic, chemically-conjugated
proteins such as Ag85A to the immune system, as VLPs are recognised readily by the
immune system and are very effective as candidate vaccines (Noad & Roy, 2003).
Rabbit haemorrhagic disease virus (RHDV), also known as rabbit calicivirus (RCV), is the
type species of the genus Lagovirus belonging to Caliciviridae family. The RHD virion is 30–
40 nm in diameter, shows a characteristic morphology, and the capsid is composed of a major
protein of 60 kDa (VP60). The 7.5 kb positive-sense ssRNA genome encodes a large
precursor polyprotein which undergoes proteolytic cleavage to yield the mature protein. The
C-terminal region of the polyprotein gives rise to the p60 polypeptide, which is detected by
antibodies against the capsid protein. This polypeptide produced from the cloned gene in
vitro, spontaneously forms VLP. The production of VLPs start with infecting insect cells
using recombinant baculovirus, and culturing the cells. Post culture, the supernatants and
lysates are separated and purified usually by sucrose gradient centrifugation, and examined
for recombinant VLPs by SDS-PAGE and immunoblotting techniques (Gromadzka et al.,
2006).
19
Antigen 85 complex
The MTB cell wall is made up of mycolic acid, arabinogalactan and peptidoglycan linked
covalently. Some of the enzymes playing a vital role in biosynthesis of the mycobacterial cell
wall are three dominant exported fibronectin-binding proteins, consisting of Antigen 85
complex. The Antigen 85 complex (Ag85A) from MTB consists of three well enough
secreted proteins (FbpA, FbpB and FbpC2) which play a key role in the pathogenesis of
tuberculosis (Kremer et al., 2002).
To determine whether Ag 85A could induce antigen-specific cellular immune response IFN-γ
production was examined in immunised mice. At one month after immunization, the spleen
cells from mice immunized with Ag 85A or BCG could produce IFN-γ in response to Ag
85A . IFN-γ level in response to Ag 85A was four times higher in Ag 85A-immunized than in
BCG-vaccinated mice (Naito et al., 1999).
MV85A (Modified vaccinia virus Ankara expressing antigen 85A)
Vaccinia virus can be genetically modified to become carriers tha t produce recombinant
proteins. Vaccinia viruses are reconstructed to express foreign genes, and are used as vectors
(vaccine candidates) to deliver antigens.
MV85A is a recombinant modified vaccinia virus Ankara, that expresses the antigen 85A
derived from mycobacteria. It was found to induce high levels of antigen-specific IFN-γsecreting T cells when used alone in bacille Calmette-Guérin (BCG)-naive healthy
volunteers. In these volunteers who had been vaccinated 0.5−38 years previously with BCG,
substantially higher levels of antigen-specific IFN-γ-secreting T cells were induced. At 24
weeks after vaccination these levels were 5−30 times greater than in vaccines administered
simply a single BCG vaccination. Therefore MVA85A could be used clinically to boost the
capacity of the standard BCG vaccine. (McShane et al., 2004)
20
1.7 Objectives of this study

To establish base- line responses in BCG- vaccinated mice using lymphocyte
transformation and cytokine assays.

To produce RHDV VLPs and chemically couple Ag85A peptide from Mycobacterium
bovis to them.

To evaluate the effects of exposure to environmental mycobacteria on BCG
vaccination as well as on MVA/Ag85A and VLP/Ag85A vaccines by orally presensitising mice with WAg206 prior to vaccination.
21
Chapter-2
MATERIALS
&
METHODS
22
2.1 Animals
Specific pathogen free (SPF) BALB/ c mice were obtained from the Department of Animal
Laboratory sciences, University of Otago, Dunedin. All the experiments using mice were
covered by a permit from the Otago University Animal Ethics Committee (AEC No: 51/08).
2.2 Media
Culture medium comprised Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen
corporation) + 5% (vol.), Foetal Calf Serum (FCS) + 0.1% (vol.), 2-Mercaptoethanol
(Invitrogen corporation) (net conc. at 55mM in D-PBS) + 5% (vol.), PenStrep (Penicillin at
10000 units/ml, Streptomycin at 10,000 µg/mg) (Invitrogen corporation).
2.3 Novel Vaccines used
2.3.1 VLP-Ag85A pre paration
RHDV VLP preparation was initiated by infecting Sf-21 cells (Spodopftera frugiperda IPLBsf21) (400 ml; 106 cells/ml) with recombinant baculovirus containing the VP60 gene in
medium supplemented with PenStrep (20 U (12µg) Pencillin / 20 µg Streptomycin). An MOI
of 1 was used. The cell count was calculated using haemocytometer and plaque-assayed viral
inoculum. The culture was incubated for 4 days at 28 o C with shaking followed by treatment
with 2 ml (0.05%) TritonX-100 detergent (SIGMA, MO, USA) for 1 hour at RT. The culture
was then centrifuged at 10,000g for 20min in a JA14 rotor (Beckman Coulter, CA, USA)
with the supernatant being transferred to Ti70 rotor (Beckman Coulter, CA, USA). After
centrifuging for 90 min at 100,000 g, the pelleted VLP was re-suspended overnight (at 4 0 C in
500µl coupling PBS) (0.26% NaH2 PO 4 .2H2 O + 1.18% Na2 PO4 + 0.87% NaCl in distilled
water, PH at 7.2)after discarding the supernatant.
23
Residual debris was removed from the re-suspended pellets by centrifuging them at 10,000g
for 20 minutes, and then the supernatant were collected and loaded onto 3 ml of caesium
chloride (CsCl at 1.2g/cm3 ) and then further overlayed with 3ml of CsCl at 1.4 g/cm3 in
SW32.1 tubes. The tubes were then topped up with insect PBS and then subjected to
centrifugation at 100,000g at 4 0 C for 18 hours using a SW 32 Ti rotor (Beckman Coulter).
The translucent band produced on the gradient was harvested using a Pasteur pipette over a
light box. SDS-PAGE was used to confirm the presence of RHDV VLP, and the sample was
dialysed for 2 hours twice using Slide-A-Lyzer dialysis cassette (Pierce technologies, Aalst,
Belgium), and then finally overnight. The VLP production for this work was carried out by
Stephanie Win with help from Ms. Vivian Young, Dept of Microbiology, University of
Otago.
Sulfo-SMCC (No.22322, Pierce, Illinois) are often used to prepare antibody- enzyme and
hapten-carrier protein conjugates in a two step reaction scheme. First, the amine-containing
protein is reacted with a several fold molar excess of cross linker followed by removal of
excess (non reacted) reagent by desalting or dialysis and, finally, the sulfhydryl-containing
molecule is added to react with the melamide groups already attached to the first protein.
The procedure carried out for this project was as follows.
Vaccine of volume 1.7 mg was prepared (1.5mg for inoculation (in vivo)) by chemical
conjugation technique.
SMCC (Sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) was taken at
20 times molar excess of 1ml of RHDV VLP at 5.5mg/ml (9.17 x 10-8 moles). So the
molarity of Sulfo-SMCC (436.37 g/mol) was approximately 1.8 X 10-6 moles. When
converted to weight, it was about 0.8mg of SMCC. 10µl of Dimethyl formamide (DMF) was
added to 0.8mg of sulfo-SMCC.
24
0.8mg of Sulfo-SMCC was added to 1ml of VLP (conc. at 5.5mg/ml) and incubated at room
temperature with shaking (rotation) for 30 minutes (total volume 1.5 ml).
Two hundred times the sample volume (300ml) of PBS (Phosphate buffered solution with
NaCl) (0.26% NaH2 PO4 .2H2O + 1.18% Na2 HPO 4 + 0.87% NaCl in Distilled water, P H
7.2)was prepared and used to dialyse the sample. The sample was dialysed twice for 2 hours,
and then a third time overnight.
Sulfo-SMCC-VP60 was harvested from Slide-A-Lyzer dialysis cassette (Pierce technologies,
Aalst, Belgium) using a syringe, and 5mg of Sulfo-SMCC-VP60 was retained after dialysis.
Ag85A CD4 (Auspep) peptide molecule was used as the antigen to couple to the VLP. To
couple Ag85A (2648 g/mol) with Sulfo-SMCC-VP60 Ag85A peptide was prepared at 10
times molar excess of Sulfo-SMCC-VP60, therefore 8.3 X 10-7 moles of Ag85A (2.2mg
peptide) was added to Sulfo-SMCC-VP60. This was incubated at room temperature for 1
hour. The sample was again dialysed twice for 2 hours, and then third time overnight.
VP60-Sulfo-SMCC-Ag85A was again harvested from Slide-A-Lyzer dialysis cassette (Pierce
technologies, Aalst, Belgium) and SDS-PAGE was performed to confirm the coupling.
SDS-PAGE (Polyacrylamide gel electrophoresis)
All samples (VP60, VLP/Ag85A, BSA) were diluted in equal volume of 2 X SDS-PAGE
sample buffer and boiled for 5 minutes prior to loading o n 10% acrylamide gels. For a
Coomassie stained gel Broad Range ladder (New England Biolabs, Ipswich, UK) was used.
The gel was run at 170 V until the dye front had reached the end of gel in a gel tank (Biorad,
CA, USA). The gels were then removed and stained with Coomassie blue for 30 minutes and
then placed in destain (10% Methanol + 10% Acetic acid in distilled water) with tissue paper
to remove all unwanted stain. Protein bands were captured using a ChemiDoc system
(Biorad).
25
2.3.2 MVA/AG85A
The MVA/AG85A vaccine used in this study was obtained as a gift from Oxford University
(Dr Sarah Gilbert).
2.4 Vaccination schedule
The mice were divided into 6 groups based on the vaccination and are tabulated as follows:
TABLE 2. Vaccination strategy in different mice groups used in the study.
Group
Number
Treatment
Quantity (per mouse)
Stage-1 (Preliminary experiments)
1
3
PBS
100 µl subcutaneous
2
3
BCG
106 CFU subcutaneous
Stage-2 (Pre-sensitisation experiments)
1
3
PBS
100 µl subcutaneous
2
3
BCG
106 CFU subcutaneous
106 CFU orally
(WAg206)
+
3
3
BCG + WAg206
BCG
(106 CFU
subcutaneous)
26
Stage-3 (Final experiment)
Group
Number
Treatment
Quantity (per mouse)
1
6
PBS
100 µl subcutaneous
2
6
BCG
106 CFU subcutaneous
106 CFU orally
(WAg206)
3
6
BCG + WAg206
+
BCG
(106 CFU
subcutaneous)
106 CFU orally
(WAg206)
+
4
6
VLP + WAg206
VP60
(100 µl @ 1 mg/ml)
(subcutaneous)
106 CFU orally
(WAg206)
+
5
6
VLP/Ag85A + WAg206
VLP/Ag85
(100 µl @ 1 mg/ml)
(subcutaneous)
27
Group
Number
Treatment
Quantity (per mouse)
106 CFU orally
(WAg206)
6
6
MVA/Ag85A + WAg206
+
MVA/Ag85A
(5 x 107 pfu into ear)
All the M. Avium WAg206 pre-sensitisation mice groups were orally given 10 6 CFU
WAg206 (diluted in 7H9 media). The mice were administered with respective vaccines eight
weeks after pre-sensitisation (Week 8).
Four weeks after immunisation (Week 12) in stage-3 (Final experiment), Groups 4, 5 and 6
were boosted (same quantity as before) with either VP60 or VLP/Ag85 or MVA/Ag85A
accordingly.
Four weeks later (Week 16), the mice were assessed for immunological responses to
mycobacterial antigens.
2.5 Splenocyte suspension preparation
The whole spleen was isolated from the euthanized mice (cervical dislocation). The spleen
was then transferred on to a 10ml Petri dish containing 5ml media and macerated using 5ml
syringe rubber. Clumps were removed using cell strainer under a sterile environment. The
cell suspension was increased to a volume of 10ml by topping up with media (DMEM + 5%
FCS). The cell suspension was centrifuged at 300x g and supernatant was removed. The cells
were re-suspended with media in 20ml FALCON tubes (BD, USA). The cells were counted
28
using Coulter-Counter, and then the cell suspension was diluted to a concentration of 2 X 106
cells/ml using media.
2.6 Splenocyte culture for cytokine data
Twenty four well flat bottom plates (Nunc A/S, Roskilde, Denmark) were used to culture
splenocytes for measuring the concentrations of cytokine produced by the cells with different
antigens used in our study. Within the same plates, cells were distributed evenly across wells
using pipettes for different antigens and controls: media only (negative control), ConA
(positive control)(2.5 µg/ml), PPD-b (33 µg/ml), PPD-a (33 µg/ml) and Ag85A (20 µg/ml).
One millilitre of cell suspension was added to each well, and 500µl of antigen was added to
respective wells, making the total volume to 1.5ml
Two different plates were prepared for each group of mice to examine two different
cytokines: IL-2 at 24 hour time point, and IL-5 and IFN-γ at 72 hours.
The plates were then cultured in an incubator at 37o C, 10% CO 2 . Supernatants (1.2ml
approximately) were removed at appropriate time-points and carefully transferred in to sterile
eppendorf tubes. These were spun at 800 x g for 5 minutes in order to remove debris, then
transferred to new eppendorf tubes, and frozen (at -80o C) until the time of analysis.
2.7 Splenocyte proliferation assay
Ninety six well round bottom plates (Nunc A/S, Roskilde, Denmark) were used to carry out
the proliferation assay of splenocytes. The antigens (50µl volume) were added to the wells.
Cell suspension was added at concentration of 2 x 10 6 cells/ml and the plates were incubated
at 37o C, 10% CO 2 (Sanyo CO 2 incubator Model: 17AIC) for 72 hours. The plates were taken
out to add 1 µCi of [methyl-3H]thymidine to each well diluted in 50 µl media. The cells
29
were then incubated overnight at 37 o C, 10% CO 2 before harvesting the cells with a Tomtec
HarvesterTM 96 (Model: Mach III M). The cells were harvested onto a filter mat printed with
grids, and the filter mat was dried using a microwave oven for 150 seconds.
These were placed in plastic bags, with 5ml liquid scintillant (BetaPlate ScintTM, Wallac)
added on to filter mat just before sealing. These were transferred onto cassettes of the liquid
scintillation counter(1450 Microbeta Plus Scintillation CounterTM, Wallac), and counted.
The results were obtained on the computer using the software Microbeta windows
workstation.
2.8 IFN-γ/ IL-2/ IL-5 ELISA of splenocyte culture supernatants
Nunc-ImmunoTM 96 MicrowellT M plates (Nunc A/S, Roskilde, Denmark) were incubated
overnight at 4 o C with 100 µl of anti- mouse cytokine capture antibody (BD Biosciences
Pharmingen) diluted 1:250 in ELISA Coating buffer (0.1M NaHCO 3 ). Wells were washed 46 times with ELISA wash buffer (PBS + 0.05% Tween-20) and incubated with 200µl of
blocking buffer (PBS + 1% Bovine serum albumin) for 2 hours at room temperature to block
non-specific protein binding. After blocking, the plates were washed 6 times with ELISA
Wash buffer. Then 100 µl of appropriate samples (thawed supernatants) and doubling
dilutions of the appropriate recombinant cytokine standard (BD Biosciences, Pharmingen)
was added. Plates were sealed and incubated at 4 o C overnight, then washed 6 times with
wash buffer. Plates were incubated for 1hour at room temperature with 100µl of biotinylated
anti- mouse cytokine detection antibody (BD Biosciences, Pharmingen) diluted 1:250 in
blocking buffer. Plates were washed 6 times again in wash buffer. Streptavidin- horseradish
preoxidase(HRP) conjugate enzyme(BD Biosciences Pharmingen) diluted 1:3000 in blocking
buffer was added to each well and incubated for 30 minutes at RT. Plates were washed 6
times with ELISA wash buffer and 100µl of TMB substrate reagent (BD Biosciences
Pharmingen) was added to develop the colour reaction.
30
Plates were left until a clear gradient of colour was observed across the recombinant cytokine
standard dilutions, at which point 100 µl of 1N H2 SO4 was added to stop the reaction. After
stopping the reactions, the absorbance of each well was read on Microplate reader (BIORAD
Model:550) at 450nm which converted absorbance values to the quantity of cytokine in pg/ml
using the standard curve plotted from the recombinant cytokine standard dilutions using the
software Microplate manager(ver.4.2.1, BIORAD). The duplicated samples were averaged to
give a mean measurement of cytokine production for each sample.
2.9 Total serum antibody ELISA (PPD-b / Ag85A)
Blood extracted from the mice by cardiac puncture (and allowed to clot), was used to provide
the serum.
The plates were coated with 50ul of antigen [PPD-b (Biocor) at 50 µg/ml and Ag85A (H –
Cys - Leu - Thr - Ser - Glu - Leu - Pro - Gly - Trp - Leu - Gln - Ala - Asn - Arg - His - Val Lys - Pro - Thr - Gly - Ser – NH2 ) (Auspep) at 20 µg/ml] diluted in coating buffer (0.1M
NaHCO3) and incubated at 4 0 C overnight. Plates were washed 6 times in wash buffer and
then blocked with 200ul of blocking buffer, incubated for 2 hours at room temperature. Plates
were washed again with wash buffer (0.05% Tween-20 in PBS), and 100 µl aliquots of serum
samples were added to wells in doubling dilutions starting at 1:20 diluted in blocking buffer,
and incubated for 1 hour at room temperature.
Plates were washed again with wash buffer 6 times, and 100 µl of goat anti- mouse IgG HRP
conjugate diluted 1:5000 in blocking buffer was added to each well, and the plates were
incubated for 45 minutes at room temperature.
Plates were washed 6 times, and 100 µl per well of TMB substrate was added to develop the
reaction. When colour developed, 100 µl of 1N H2 SO4 was added to each well to stop the
31
reaction, and the plates were read on Microplate reader (BIORAD Model: 550) at 450nm and
converted to Optical density values (O.D) values for the relevant amount of antibody present
in the serum.
2.10 Isotyping Serum antibody ELISA (PPD-b / Ag85A)
Plates were coated with 50ul per well of antigens (PPD-b at 50ug/ml and Ag85A at 20ug/ml)
diluted in coating buffer(0.1M NaHCO3) on separate plates, and doubling dilutions of
standards (100ul per well), with doubling dilutions starting at 500ng/ml for IgG1, and
250ng/ml for IgG2a) were added to appropriate wells. There were four different sets of plates
(IgG1-PPD-b, IgG1-Ag85A, IgG2a-PPD-b, IgG2a-Ag85A).
The plates were incubated at 4 0 C overnight and washed 6 times with wash buffer. The plates
were blocked with 200 µl per well of blocking buffer for 2 hours at room temperature. One
hundred µl per well of serum samples diluted in blocking buffer was added to samplecontaining wells, and 100ul per well of blocking buffer was added to wells containing
standards in doubling dilutions starting at 1:20 dilution. The plates were incubated for 2 hours
at room temperature.
Plates were washed 6 times with wash buffer, and 100ul of biotinylated anti- mouse
antibodies to IgG1 or IgG2a (IgG1 antibody diluted 1:8000, and IgG2a at 1:4000 in blocking
buffer) was added to wells, and the plates were incubated for 45 minutes at room
temperature. The plates were washed 6 times with wash buffer, and 100ul of StreptavidinHRP (Zymed) diluted 1:3000 in blocking buffer was added to wells, and incubated for 30
minutes at room temperature.
Plates were washed for 6 times, and developed with TMB as described previously.
32
2.11 Statistical Analysis
For statistical analysis paired t tests were used for comparison between two groups and Oneway ANOVA was used to compare whole data sets, generated by PRISM (version 5.0)
software. P Values less than 0.05 were considered significant.
33
Chapter 3
RESULTS
34
3.1 Preliminary experiments demonstrating that BCG vaccination induces immune responses to
bovine-PPD and Ag85A in mice:
Proliferation responses:
The proliferation of splenocytes was greatest when challenged with PPD-b at 33 µg/ml and Ag85A at
20ug/ml (Figure 3.1). There was a large difference between naive mice and BCG vaccinated mice
with regard to responses at both PPD-b concentrations showing that BCG vaccination elicited immune
response specific to PPD-b, and T-cells had become activated. A smaller but distinct response was
elicited to Ag85A. Since antigen-specific proliferation was detected in vaccinated mice it was of
interest to investigate IL-2 production.
Cytokine responses:
Splenocytes from BCG Vaccinated mice produce low levels of antigen-specific
IL-2 compared with naive mice where IL-2 was undetectable.
Splenocytes from naive mice produced almost no IL-2 to re-stimulation with antigen therefore low
IL-2 levels produced by splenocytes from BCG vaccinated mice were considered a positive result
(Figure 3.2). Based on the repeated results (results from one of three experiments are shown) IL-2
production was highest at PPD-b concentration of 33 µg/ml. The dose/response assay failed to detect
differences in response to three different concentrations.
Since it was established that the BCG sensitised splenocytes could produce IL-2 in response to either
PPD-b or Ag85A after 24 hours, their ability to express IFN-γ after 72 hours was investigated.
35
BCG Vaccinated mice produce antigen-specific IFN-γ but this is undetectable in naive mice.
Repeating the trend, naive mice did not produce IFN-γ in response to any of the antigens, whereas
BCG vaccinated mice produced increasing levels of IFN-γ with increasing dose of the challenge
antigens (Figure 3.3). Again, IFN-γ production was highest at PPD-b concentration of 33µg/ml. No
difference was detectable at all Ag85A concentrations where very low levels of IFN-γ were
produced.
Splenocytes from BCG vaccinated mice were shown to produce Th-1 type cytokines IL-2 and IFN-γ
in response to PPD-b and Ag85A. It was of interest to compare this with Th2 cytokines. So, the IL-5
response, representative of a Th2 response, was investigated.
No difference exists between naive mice and BCG vaccinated with regard to IL-5 production in
response to mycobacterial antigen
Splenocytes from both naive mice and BCG vaccinated mice produced very low, almost undetectable,
amounts of IL-5 to all concentrations of antigen. There was no significant difference between these
groups in response to any of the antigen after 72 hours (Figure 3.4).
To determine whether this weak Th2 response correlated with antibody antigen-specific IgG in serum
of vaccinated mice was investigated.
Antibody responses:
No difference in serum antibody levels in response to PPD-b and Ag85 between naive mice and
BCG vaccinated mice.
There was no detectable difference in the serum antibody levels in response to the antigens PPD-b and
Ag85A (Figure 3.5). This correlated with the IL-5 production suggesting that there was almost no Th2
cytokine response in BCG vaccinated.
36
Naive (Control) mice
BCG Vaccinated mice
15000
cpm
10000
5000
l
33
g
-b
/m
@
l
PP
16
D
g
-b
/m
@
l
16
A
g8
g
5
/m
@
l
20
A
g8
g
5
/m
@
l
20
A
g8
g
5
/m
@
l
10
A
g8
g
5
/m
@
l
10
g
/m
l
/m
n
@
PP
D
-b
PP
D
-b
@
33
g
nt
ig
e
A
PP
D
N
o
N
o
A
nt
ig
e
n
0
FIGURE 3.1 Comparison of proliferation of splenocytes from BCG vaccinated mice and naïve
(control) mice with different antigens (PPD-b, Ag85A). Data shown is mean ± standard error, results
from two mice in each group.
37
BCG Vaccinated mice
Conc. of IL-2 (pg/ml)
300
200
100
l
20
g
g8
/m
5
@
ll
10
A
g
g8
/m
5
l
@
5
g/
m
l
A
5
g8
A
PP
D
-b
@
@
8
g
/m
l
/m
l
16
g
/m
@
-b
D
PP
PP
D
-b
N
@
o
A
nt
33
g
ig
en
0
FIGURE 3.2 IL-2 expression (Cytokine ELISA) produced by cultured splenocytes from BCG
vaccinated mice and naïve (control) mice in response to in vitro challenge with with antigens PPD-b
and Ag85A at different concentrations. No cytokine was detected in the naive mice to any of the
antigens. Data shown is mean ± standard error, results from two mice in each group.
38
BCG Vaccinated mice
Conc. of IFN- (pg/ml)
15000
10000
5000
l
17
µg
-b
/m
@
l
A
8µ
g8
g/
5
m
@
l
20
A
g8
µg
5
/m
@
l
10
A
µg
g8
/m
5
@
l
5µ
g/
m
l
PP
D
PP
D
-b
@
D
-b
@
33
µg
nt
A
o
PP
N
/m
ig
en
0
FIGURE 3.3 Comparison of IFN-γ expression (Cytokine ELISA) produced by cultured splenocytes
from BCG vaccinated mice in response to challenge with different antigens PPD-b, Ag85A at
different concentrations. No cytokine was detected in the naive mice to any of the antigens. Data
shown is mean ± standard error, results from two mice in each group.
39
Naive
BCG vaccinated mice
Conc. of IL-5 (pg/ml)
8
6
4
2
2
m
l
17
µ
Dg/
b
m
@
l
A
8
g8
µg
5
/m
@
l
2
A
0µ
g8
g/
5
m
@
l
10
A
µ
g8
g/
5
m
@
l
5µ
g/
m
l
a
PP
PP
Db
@
33
µg
/
ed
i
Db
@
m
m
l
17
µg
D/m
b
@
l
A
8µ
g8
g/
5
m
@
l
20
A
g8
µg
5
/m
@
l
1
0µ
A
g8
g/
5
m
@
l
5µ
g/
m
l
PP
PP
Db
@
33
µg
/
ed
i
@
m
Db
PP
4
0
a
0
6
PP
Conc. of IL-5 (pg/ml)
8
FIGURE 3.4 Comparison of IL-5 expression produced by cultured splenocytes from BCG vaccinated
mice versus naïve mice in response to challenge with different antigens PPD-b, Ag85A at different
concentrations. Data shown is mean ± standard error, results from two mice in each group.
40
NAIVE
1.5
BCG
O.D
1.0
0.5
0.0
10
20
40
80 160
10
20
40
80 160
Reciprocal of Serum dilution
FIGURE 3.5 Comparison of Serum antibody levels to PPD-b obtained from BCG vaccinated mice
group and naïve (control) mice.
41
3.2 Pre-sensitising mice by oral administration of M. Avium WAg206 affects the immune
response to BCG in mice
Proliferation responses:
Splenocytes from BCG vaccinated mice re-challenged with PPD-b mycobacterial antigen in vitro
recorded higher proliferation than BCG vaccinated mice exposed to M. avium WAg206 (Fig 3.6).
This trend was reversed when splenocytes were challenged with PPD-a in vitro. No responses to
Ag85A were detected either from BCG vaccinated mice or BCG vaccinated mice exposed to M.
avium WAg206. It was of interest to determine whether these results were reflected in IL-2
production.
Cytokine responses
Modulation of IL-2 production to PPD-b by oral pre-sensitisation with M. Avium WAg 206
Prior exposure to M. avium strain WAg 206 resulted in a trend towards decreased levels of IL-2 in
BCG immunised mice in response to PPD-b, but increased levels of IL-2 in response to PPD-a (Figure
3.7). In response to Ag85A, M. avium WAg 206 presensitised group produced (statistically
significant) more IL-2, though the difference was small.
Since differences in IL-2 production were detectable in pre-sensitised mice, it was of interest to
investigate whether these were reflected in IFN-γ production.
42
Modulation of IFN-γ production:
Exposure to M. avium strain WAg 206 prior to immunisation with BCG resulted in decreased levels
of IFN-γ following challenge with PPD-b, PPD-a and Ag85A, and increased levels of IFN-γ in
response to PPD-a (P < 0.05) (Figure 3.8). This was in contrast to IL-2 production where presensitisation with WAg206 resulted in an increase of this cytokine when the splenocytes were
challenged with Ag85A. Since this results suggested that the predominant Th1 response to PPD-b and
Ag85A was reduced by WAg206 pre-sensitisation, the Th2 response exemplified by IL-5 production
was investigated.
Modulation of IL-5 production:
Though the pre-sensitisation with WAg206 made no difference to IL-5 production, it did result in
increased responses to PPD-a and Ag85A (P < 0.05) (Figure 3.9). However the actual amounts of IL-5
produced in all the groups were very low, suggesting that there was a limited Th2 type response.
Antibody responses:
There was no difference observed between the groups with regard to total serum antibody levels
specific to PPD-b (Figure 3.10(a)), all the levels being very low.
Levels of IgG1 antibody specific to PPD-b were lower in M. avium pre-sensitised BCG vaccinated
mice than in BCG vaccinated mice but this was not staististically significant. It was opposite in the
case of IgG1 antibody specific to PPD-a (Figure 3.10(b)), though again the results were not
statistically significant. Low levels (but statistically significant) of IgG2a antibody specific to antigens
PPD-b were produced in all the groups, and showing that more antibody produced by M. avium presensitised BCG vaccinated mice when compared to BCG vaccinated mice.
43
**
15000
Media only
PPD-bovine
PPD-avium
Ag85
cpm
10000
5000
Na
iv
e
BC
W
G
Ag
on
20
ly
6
+
BC
G
Na
iv
e
BC
W
G
Ag
on
20
ly
6
+
BC
G
Na
iv
e
BC
W
G
Ag
on
20
ly
6
+
BC
G
Na
iv
e
BC
W
G
Ag
on
20
ly
6
+
BC
G
0
FIGURE 3.6 Comparison of proliferation of splenocytes from BCG vaccinated mice, BCG vaccinated
mice presensitised orally with M. avium strain WAg206 and naïve (control) mice in response to PPDb, PPD-a and Ag85A. ** p < 0.005, compared to cells stimulated with BCG alone. Data shown is
mean ± standard error, results from three mice in each group.
44
Conc. of IL-2 (pg/ml)
250
Media only
PPD-bovine
PPD-avium
Ag85
200
150
*
100
50
Na
iv
e
BC
W
G
Ag
on
20
ly
6
+
BC
G
Na
iv
e
BC
W
G
Ag
on
20
ly
6
+
BC
G
Na
iv
e
BC
W
G
Ag
on
20
ly
6
+
BC
G
Na
iv
e
BC
W
G
Ag
on
20
ly
6
+
BC
G
0
FIGURE 3.7 Comparison of IL-2 expression produced by cultured splenocytes from BCG
vaccinated mice , BCG vaccinated mice pre-sensitised orally with M. avium strain WAg206
and naïve (control) mice in response to different antigens (PPD-b, PPD-a, Ag85A). * p <
0.05, BCG vaccinated mice pre-sensitised orally with M. avium strain WAg206 compared to
cells stimulated with BCG alone. Data shown is mean ± standard error, results from three
mice in each group. This result is representative of two repeat experiments.
45
*
**
10000
5000
***
Na
iv
e
BC
W
G
Ag
on
20
ly
6
+
BC
G
Na
iv
e
BC
W
G
Ag
on
20
ly
6
+
BC
G
Na
iv
e
BC
W
G
Ag
on
20
ly
6
+
BC
G
1000
800
600
400
200
0
Na
iv
e
BC
W
G
Ag
on
20
ly
6
+
BC
G
Conc. of IFN- (pg/ml)
15000
FIGURE 3.8 Comparison of IFN-γ expression (cytokine ELISA) produced by cultured splenocytes
from BCG vaccinated mice, BCG vaccinated mice pre-sensitised orally with M. avium strain
WAg206 and naïve (control) mice in response to different antigens (PPD-b, PPD-a, Ag85A). * p <
0.05, ** p<0.005, *** p < 0.0005, Data shown are mean ± standard error; results from three mice in
each group. This result is representative of two repeat experiments.
46
Media only
PPD-bovine
PPD-avium
Ag85
Conc. of IL-5 (pg/ml)
40
*
30
*
20
10
Na
ive
BC
W
G
Ag
on
20
ly
6+
BC
G
Na
ive
BC
W
G
Ag
on
20
ly
6+
BC
G
Na
ive
BC
W
G
Ag
on
20
ly
6+
BC
G
Na
ive
BC
W
G
Ag
on
20
ly
6+
BC
G
0
FIGURE 3.9 Comparison of IL-5 production by cultured splenocytes from BCG vaccinated
mice , BCG vaccinated mice pre-sensitised orally with M. avium strain WAg206 and naïve
(control) mice in response to different antigens (PPD-b, PPD-a, Ag85A)(* p < 0.05). Data
shown is mean ± standard error, results from three mice in each group. This result is
representative of two repeat experiments.
47
Media only
PPD-bovine
PPD-avium
Ag85
a.
PPD-b
1.0
O.D
0.8
0.6
0.4
0.2
G
BC
W
A
B
g2
06
+
CG
N
on
ly
ai
ve
0.0
Reciprocal of Serum dilution
* serum dilution @ 1/20
b.
PPD-bovine (IgG1)
IgG1
PPD-bovine (IgG2a)
PPD-avium (IgG1)
IgG2a
PPD-avium (IgG2a)
500
Conc. of IgG1 (ng/ml)
3000
2000
1000
400
*
300
200
100
G
ly
BC
+
iv
e
on
6
Ag
20
W
Na
BC
G
G
BC
ly
+
on
6
Ag
20
W
Na
G
Ag
on
ly
20
6
+
BC
G
BC
W
W
Na
iv
e
Ag
on
20
ly
6
+
BC
G
G
Na
iv
e
BC
iv
e
0
0
BC
G
Conc. of IgG1 (ng/ml)
4000
FIGURE 3.10 (a). Comparison of Serum total-antibody levels specific to PPD-b obtained from BCG
vaccinated mice, BCG vaccinated mice pre-sensitised orally with M. avium strain WAg206 and naïve
(control) mice in response to PPD-b and PPD-a. Data shown is mean ± standard error, results from
three mice in each group).
(b). Comparison between levels of PPD-b specific IgG1, IgG2a antibody response and PPD-a
specific IgG1, IgG2a antibodies in sera obtained from BCG vaccinated mice , BCG vaccinated mice
pre-sensitised orally with M. avium strain WAg206 and naïve (control) mice in response to PPD-b
and PPD-a ( *p < 0.05).
48
3.3 VLP can be coupled with Ag85A using a che mical conjugation technique
VLP (VP60) peptide was chemically conjugated with Ag85AA peptide using Sulfo-SMCC, a
hetero bi- functional linker, as described in Materials and Methods. VLP conjugated with
Ag85A (VLP/Ag85A) was analysed on a standard 10% SDS-PAGE gel before being stained
with Coomassie blue and pictured using a Chemidoc. Figure 3.11 shows the well defined
bands of VLP/Ag85A (at around 65 kDa) compared to VP60 (VLP only) peptide (around 60
kDa). This confirmation of coupling was essential to establish that this novel vaccination
candidate VLP/Ag85A was ready for the test to check its efficacy as a novel vaccine
candidate against TB.
49
Figure 3.11 Chemically conjugated VLP/Ag85AA run on a 10% SDS-PAGE gel along with
VP60 (VLP only) and titrations of BSA used as positive control. A Broad range marker (New
England Biolabs) was used to assign VLP/Ag85A size.
50
3.4 Vaccination with VLP/Ag85A generated low antigen-specific IFN-γ response only in mice.
Proliferative responses:
Surprisingly, the splenocytes from VP60 vaccinated mice had higher proliferation than
VLP/Ag85A vaccinated mice under all conditions (sensitised in vitro with antigens PPD-b,
PPD-a and Ag85A) and the responses were statistically significant (Figure 3.12).
Cytokine responses:
No IL-2 was detected in response to any of the antigens PPD-b, PPD-a and Ag85A with any
of the vaccinated groups (naive, VP60 and VLP/Ag85A) (data not shown).
Very low levels of IFN-γ that were almost negligible were detected in response to the
antigens PPD-b, PPD-a and Ag85A in both the vaccinated groups (VP60 and VLP/Ag85A)
(Figure 3.13). Significantly more IFN-γ was detected to PPD-b and PPD-a in mice vaccinated
with VLP/Ag85A.
As the splenocytes were shown to produce Th-1 type cytokines IL-2 and Interferon-γ in
response to PPD-b and Ag85A, IL-5 response (Th2 response) was investigated. No IL-5
levels were detected in any of the groups.
51
*
2500
VP60
2000
cpm
VLP/Ag85A
1500
1000
*
500
g8
5
A
g8
5
A
N
N
o
A
n
o tige
A
nt n
ig
en
P
P
D
P bo
P v
D in
-b e
ov
in
P
e
P
D
P av
P iu
D
-a m
vi
um
0
FIGURE 3.12 Comparison of proliferation of splenocytes from VLP alone (VP60)
administered mice and VLP/Ag85A vaccinated mice in response to PPD-b, PPD-a and
Ag85A (** p < 0.005, VLP/Ag85A vaccinated mice compared to VLP alone (VP60)
administered mice. Data shown is mean ± standard error; results from six mice in each
group).
52
VP60
**
VLP/Ag85A
30
**
20
10
N
g8
5
A
o
A
g8
5
0
A
N nti
o ge
A
nt n
ig
en
P
P
D
P bo
P v
D in
-b e
ov
in
P
e
P
D
P av
P iu
D
-a m
vi
um
Conc. of IFN- (pg/ml)
40
Figure 3.13 Comparison of IFN-γ produced by cultured splenocytes from VP60(VLP only)vaccinated mice and VLP/Ag85A- vaccinated mice in response to antigens PPD-b, PPD-a and
Ag85A. ** p < 0.005, VLP/Ag85A vaccinated mice compared to VLP alone (VP60)
administered mice. Data shown is mean ± standard error; results from six mice in each group.
53
3.5 Pre-senstising mice with M. avium WAg206 has less effect on antigen-specific
immune response to MVA85A than on those to VLP/Ag85A
Proliferation responses:
When all groups were exposed to M. avium strain WAg206 prior to vaccination, the
splenocytes from MVA85A vaccinated mice had significantly higher proliferative response to
all mycobacterial antigens PPD-b, PPD-a and Ag85A) when compared to rest of the groups
(Figure 3.14).
Interestingly, splenocytes from VLP only vaccinated mice had a higher prolifera tive response
to PPD-b than VLP/Ag85A vaccinated mice. The reverse was observed in response to Ag85A
although the degree of proliferation was low.
The splenocytes from VLP/Ag85A vaccinated mice had equal or slightly higher proliferation
than BCG vaccinated mice in response to PPD-a and Ag85A but a trend towards a lower
response to PPD-b.
Cytokine responses:
Modulation of IL-2 production:
When all mice groups were pre-sensitised with M. avium WAg 206, the mice vaccinated with
novel TB vaccine candidates VLP/Ag85A and MVA85A unexpectedly produced less IL-2 in
response to PPD-b when compared to BCG (Figure 3.15). The levels of IL-2 in all groups
were, however, low.
54
By contrast, the levels of IL-2 to PPD-a in the novel vaccination groups were higher than in
the BCG vaccinated, the levels being higher than in response to PPD-b. In addition the levels
of IL-2 to Ag85A in the novel vaccination groups were higher than in BCG vaccinated mice.
The MVA85A group had higher levels of IL-2 production when compared to VLP/Ag85A
except in response to PPD-a.
Modulation of IFN-γ production
When all vaccination groups of mice were pre-sensitised with M. avium WAg206, the levels
of IFN-γ were higher in MVA85A group than in VLP/Ag85A response to PPD-b, PPD-a and
Ag-85. The VLP/Ag85AA vaccinated group produced lower levels of IFN-γ than BCG
vaccinated mice except in response to PPD-a repeating the trend of IL-2.
Surprisingly the VP60 (VLP only) group (negative control for VLP/Ag85A) produced similar
amounts of IFN-γ when compared to VLP/Ag85A vaccinated mice except when challenged
with Ag85A (Figure 3.16).
Modulation of IL-5 production:
When all vaccination groups of mice were pre-sensitised with M. avium WAg206, only the
mice immunised with VLP/Ag85AA produced IL-5 in response to Ag85Aand even this
resulted in low levels of this cytokine (Figure 3.17).
Antibody responses:
There was no significant difference in serum antibody levels to PPD-b between vaccinated
groups pre-sensitised with M. avium WAg 206 (Fig. 3.18 (a)). The levels were low in all
55
groups. However VLP/Ag85AA vaccinated mice produced relatively higher amounts of
antibody specific to Ag85A.
IgG1 antibody isotype production was higher when compared to IgG2a, specific to PPD-b
and Ag85A.
The BCG vaccinated group of mice produced higher levels of IgG1 specific to PPD-b than any other
group, and VLP/Ag85A vaccinated mice had significantly higher levels of IgG1 and IgG2a specific to
Ag85A (Figure 3.18(b)).
No other group of vaccinated mice produced significant levels of IgG2a specific to PPD-b and
Ag85A.
56
PPD-bovine
Media
n/s
15000
15000
10000
*
***
ccpm
85
A
85
A
+
W
W
Ag
2
Ag
2
06
06
W
Ag85A
***
*
VA
/A
g
VL
06
Ag
2
06
Ag
2
W
PPD-Avium
15000
P/
Ag
VL
BC
+
PB
W
Ag
S
20
6
+
BC
W
G
Ag
W
20
Ag
6
+
20
VL
6
P
+
VL
W
Ag
P/
Ag
20
85
6
+
A
M
VA
/A
g8
5A
+
0
+
0
G
5000
PB
S
5000
P
**
M
ccpm
10000
***
ccpm
ccpm
10000
5000
15000
14000
13000
12000
11000
10000
9000
8000
4000
***
**
***
**
3000
2000
1000
0
M
W
Ag
20
6
+
+
20
6
5A
85
A
VL
20
6
VA
/A
g8
+
P/
Ag
VL
P
BC
G
+
A
g8
5
PB
S
20
6
W
Ag
W
Ag
W
Ag
06
Ag
2
W
/A
g8
5A
+
+
06
Ag
2
W
M
VA
06
VL
P/
A
+
BC
+
Ag
2
W
06
Ag
2
W
VL
P
G
PB
S
0
Figure 3.14 Comparison of proliferation of splenocytes (CPM) of different vaccination
groups (BCG, VLP only, VLP/Ag85AA, MVA85A) pre-sensitised with M. avium strain
WAg206 and PBS (control mice) in response to PPD-b, PPD-a and Ag85A (* p <0.05, ** p <
0.005, ***p<0.0005). Data shown is mean ± standard error, results from six mice in each
group.
57
Media
PPD-bovine
400
Conc. of IL-2 (pg/ml)
300
200
100
300
*
200
100
0
PB
W
Ag
S
20
6
+
BC
W
G
Ag
W
20
Ag
6
+
20
VL
6
P
+
V
W
L
Ag
P/
Ag
20
85
6
+
A
M
VA
/A
g8
5A
PB
W
Ag
S
20
6
+
BC
W
G
Ag
W
20
Ag
6
+
20
VL
6
P
+
VL
W
Ag
P/
A
20
g8
6
5A
+
M
VA
/A
g8
5A
0
PPD-Avium
Ag85A
+
06
Ag
2
W
FIGURE 3.15 Comparison of IL-2 expression in different vaccination groups (BCG, VLP
only, VLP/Ag85AA, MVA85A) pre-sensitised with M. avium strain WAg206 and PBS
(control mice) in response to PPD-b, PPD-a and Ag85A (* p <0.05, ** p < 0.005,
***p<0.0005). Data shown are mean ± standard error, results from six mice in each group.
58
A
g8
5
M
VA
/A
VL
P/
A
+
g8
5A
VL
P
G
06
+
BC
W
Ag
2
W
Ag
20
6
W
200
100
80
60
40
20
0
06
VL
P
VL
Ag
P/
A
20
g8
6
5A
+
M
VA
/A
g8
5A
+
+
W
Ag
20
6
Ag
20
6
W
Ag
20
6
+
BC
PB
S
G
0
250
S
100
*
300
PB
200
350
Ag
2
300
W
400
*
W
***
Conc. of IL-2 (pg/ml)
Conc. of IL-2 (pg/ml)
400
+
Conc. of IL-2 (pg/ml)
400
Media
PPD-bovine
15000
Conc. of IFN-(pg/ml)
12000
10000
8000
1000
800
600
400
200
0
*
10000
5000
5A
A
M
VL
P
+
+
06
06
Ag
2
Ag
2
W
W
W
Ag
VA
/A
g8
/A
g
+
06
+
Ag
2
06
W
Ag
2
W
20
6
85
VL
P
BC
G
PB
S
85
A
5A
VA
/A
g
M
+
+
VL
P
/A
g8
+
20
6
W
Ag
20
6
+
BC
G
W
Ag
20
6
W
Ag
VL
P
0
PB
S
Conc. of IFN-(pg/ml)
14000
PPD-Avium
Ag85A
*
10000
5000
0
**
14000
Conc. of IFN-(pg/ml)
12000
**
10000
8000
3000
2000
**
1000
(control mice) in response to PPD-b, PPD-a and Ag85A (* p <0.05, ** p < 0.005,
***p<0.0005). Data shown are mean ± standard error; results from six mice in each group .
59
A
M
VA
+
06
FIGURE 3.16 Comparison of IFN-γ expression in different vaccination groups (BCG, VLP
only, VLP/Ag85AA, MVA85A) pre-sensitised with M. avium strain WAg206 and PBS
/A
g8
5
g8
5A
+
W
Ag
2
W
Ag
2
06
W
Ag
2
06
VL
P/
A
+
VL
P
BC
G
06
+
PB
S
PB
W
Ag
S
20
6
+
BC
W
G
Ag
W
20
Ag
6
+
20
VL
6
P
+
VL
W
Ag
P/
Ag
20
85
6
+
A
M
VA
/A
g8
5A
0
W
Ag
2
Conc. of IFN-(pg/ml)
15000
Media
PPD-b
150
Conc. of IL-5 (pg/ml)
100
50
100
50
A
M
VA
+
+
W
Ag
20
6
6
W
Ag
20
/A
g8
5
Ag
8
VL
P
+
VL
P/
6
+
Ag
20
6
Ag
20
W
+
6
W
Ag
20
W
Ag
20
BC
G
PB
S
A
M
VA
VL
P/
+
W
/A
g8
5
5A
Ag
8
VL
P
+
6
Ag
20
6
+
BC
G
PB
S
Ag
20
W
6
Ag85A
PPD-Avium
150
Conc. of IL-5 (pg/ml)
150
100
50
(P Value < 0.05)
*
100
50
VL
Ag
P/
Ag
20
85
6
+
A
M
VA
/A
g8
5A
+
W
W
Ag
20
6
Ag
20
6
+
+
BC
G
W
PB
S
Ag
20
6
A
W
Ag
20
6
+
M
VA
VL
P/
+
/A
g8
5
5A
Ag
8
VL
P
+
6
W
Ag
20
6
Ag
20
W
6
+
BC
G
PB
S
Ag
20
W
VL
P
0
0
W
Conc. of IL-5 (pg/ml)
5A
0
0
W
Conc. of IL-5 (pg/ml)
150
Figure 3.17 Comparison of IL-5 expression in different vaccination groups (BCG, VLP only,
VLP/Ag85AA, MVA85A) pre-sensitised with M. avium strain WAg206 and PBS (control
mice) in response to PPD-b, PPD-a and Ag85A. *p < 0.05 data shown are mean ± standard
error; results from six mice in each group.
60
61
06
M
Ag
VA
/
85
A
5A
g8
W
Ag
20
6
20
6
+
+
M
85
A
VA
/A
g8
5A
P
VL
VL
P/
Ag
+
10000
+
P/
A
0
VL
500
BC
G
30000
W
Ag
2
1000
P
2000
VL
2000
+
IgG2a - PPD-b
+
2500
20
6
0
20
6
1500
W
Ag
***
***
20
6
IgG1 - PPD-b
W
Ag
10000
+
* Serum dilution @ 1/20
W
Ag
Reciprocal of Serum dilution
G
0
BC
0
PB
W
Ag
S
20
6
+
BC
W
G
Ag
W
20
Ag
6
+
20
VL
6
P
+
VL
W
Ag
P/
A
20
g8
6
5A
+
M
VA
/A
g8
5A
1
PB
S
20000
Conc. in ng/ml
1
+
A
Ag
85
A
g8
5
O.D
2
20
6
M
VA
/
VL
P
3
W
Ag
+
+
VL
P/
A
6
PB
W
Ag
S
20
6
+
BC
W
G
Ag
W
20
Ag
6
+
20
VL
6
P
+
VL
W
Ag
P/
A
20
g8
6
5A
+
M
VA
/A
g8
5A
3
PB
S
6
+
BC
G
S
4
20
6
2500
Conc. in ng/ml
W
Ag
20
6
+
PB
PPD-bovine
W
Ag
P
W
Ag
20
6
W
Ag
20
W
Ag
20
O.D
**
4
W
Ag
VL
P/
Ag
85
+
BC
G
A
VA
/A
g8
5A
VL
M
+
6
+
PB
S
Conc. in ng/ml
30000
+
6
6
W
Ag
20
W
Ag
20
6
W
Ag
20
W
Ag
20
Conc. in ng/ml
a.
Ag85
**
**
**
2
Reciprocal of Serum dilution
* Serum dilution @ 1/20
b.
IgG1 - Ag85
20000
*
*
*
1000
800
600
400
200
0
IgG2a - Ag85
*
*
1500
100
80
60
40
20
0
FIGURE 3.18 (a). Comparison of total serum antibody levels specific to PPD-b and Ag85A
through (1 in 20 dilution) of sera obtained from in different vaccination groups (BCG, VLP
only, VLP/Ag85AA, MVA85A) pre-sensitised with M. avium strain WAg206 and PBS
(control mice). * p <0.05, ** p < 0.005, ***p<0.0005 data shown is mean standard error;
results from six mice in each group.
(b). Comparison between levels of PPD-b specific IgG1, IgG2a antibodies and PPD-a
specific IgG1, IgG2a antibodies (in ng/mL) in sera obtained from in different vaccination
groups (BCG, VLP only, VLP/Ag85AA, MVA85A) pre-sensitised with M. avium strain
WAg206 and PBS (control mice).
62
3.6 Summary of results

In the preliminary experiments, splenocytes from BCG vaccinated mice had
maximum proliferation in response to PPD-b at 33ug/ml, and the cytokine assay (high
IFN-γ) supported this result, suggesting that BCG elicits good Th1 type immune
response generally.

When BCG vaccinated mice, pre-exposed to M. avium WAg206, were compared with
BCG-only vaccinated mice, the antigen-specific proliferation of splenocytes was
higher in the latter group to PPD-b, and the trend was opposite in response to PPD-a.
The same trend was seen in cytokine assays (levels of IL-2, IFN-γ). This trend was
reflected in the antigen-specific IgG1 antibody response, where levels were higher
than IgG2a.

VLP was coupled with Ag85 (by me with the help of Stephanie Win and Vivian
Young) using chemical cross- linker Sulfo-SMCC. This was confirmed by SDS-PAGE
as the VLP/Ag85 was at 65 KDa on the broad-range ladder and VP60 (VLP only) was
seen at 60 KDa.

Vaccination with VLP/Ag85A generated poor antigen-specific proliferative response,
and the same trend was seen in cytokine assays (IFN-γ) as well. No IL-2 was recorded
at all conditions. VLP-only vaccinated mice generated higher responses than
VLP/Ag85A.

In the final experiment where novel vaccination groups VLP/Ag85A and MVA85A
were pre-exposed to M. avium WAg206, MVA85A had higher proliferative responses
to all antigens than other vaccination groups. The same trend was seen in levels of IL2 and IFN-γ (cytokine assay) except IL-2 being higher in VLP/Ag85 group in
response to PPD-a. High levels of IL-5 were observed in VLP/Ag85A vaccinated
mice in response to Ag85A.
63

There was no significant difference in total antibody levels in response to PPD-b in
any of the vaccinated groups, but VLP/Ag85 vaccinated mice had higher levels in
response to Ag85.

High levels of IgG1 antibody to were recorded in mice group vaccinated with
VLP/Ag85A when compared with MVA85A vaccinated group. This suggests that
VLP/Ag85A vaccinated mice group elicited a predominant Th2 type response, which
is not protective. MVA85A vaccinated mice strong Th1 type of response (Protective)
which seems to be a better vaccine candidate.
64
Chapter-4
DISCUSSION
65
4.1 Protective immune response conferred by BCG
BCG is an attenuated live bacterial vaccine that is capable of inducing a protective immune
response against TB under normal conditions. BCG has been widely distributed worldwide
for use against TB with over three billion doses having been administered. As such it is
regarded as a safe vaccine and can be given at birth (Cassanova et al., 1996). The low
production cost makes it an ideal, cost-effective preventative measure against TB, as
compared to using antibiotics to treat disease, particularly in develop ing countries. BCG is
the best vaccine tested to date against TB (Silva et al., 1998). It is currently given
intradermally as a live, attenuated vaccine, as studies have shown that heat-killed bacteria
elicit a different and ineffectual immune response (Hook et al., 1996, Duagelat et al., 1995).
The immune response associated with protection that follows BCG vaccination, is a Th1 type
T-cell mediated immune response (Arnaud Marchant et al, 1999).
To confirm the generation of an immune response to BCG vacc ination under normal
conditions existing in our laboratory, preliminary experiments were carried out. Splenocytes
from BCG-vaccinated mice proliferated on re-stimulation with immunodominant antigens
PPD-b and Ag85A in vitro. In addition we wished to confirm the type of immune response
that was induced. As expected, BCG vaccinated mice generated good levels of IL-2 when
compared to naive mice, which produced no detectable IL-2. PPD-b-specific IL-2 production
was higher than Ag85A-specific IL-2 levels in BCG vaccinated mice. The same trend was
repeated with antigen-specific IFN-γ levels. The high levels of IFN-γ suggest that BCG elicits
strong Th1 type of immune response, as IFN-γ and IL-2 are associated with this response.
Very low levels of antigen-specific IL-5, a Th2 type of cytokine, were detected. The low
levels of PPD-specific antibody correlates with the results of Young et al following the
adminstration of BCG.
66
4.2 Modulation of immune response by BCG following exposure to environmental
mycobacteria
When BCG is efficacious it induces a Th1 immune response in the host. However the
efficacy of BCG varies greatly. Previous studies show that prior exposure of animals to
environmental mycobacteria M. avium interferes with the immune response elicited towards
BCG after vaccination (Young et al, 2007), and ultimately suppresses the protective immune
response leading to failure of BCG vaccine(Brandt et al., 2002).
As BCG is a live vaccine, it is particularly sensitive to pre-existing immune responses to
antigens that it has in common with certain strains of environmental mycobacteria. At the
antigen level, many mycobacterial species are also cross-reactive, with certain
immunodominant antigens being highly conserved between strains (Harboe et al., 1979,
Chapras et al., 1970).
Previous studies by Brandt et al (2001) show that animals exposed to several environmental
mycobacteria raise an immune response that controls the multiplication of BCG, thereby
limiting the vaccine- induced immune response before it is completely generated. These
studies hypothesised that prior exposure to environmental mycobacteria can prime host’s
immune system against shared mycobacterial antigens, and subsequently interferes with the
immune response elicited towards BCG after vaccination. In this situation the animals made a
strong Th1 immune response to the M. avium strains and thus when animals were given BCG
post M. avium infection, a strong cross-reactive Th1 immune response ensued, which
removed BCG before it could secrete any immunodominant antigens associated with priming
a protective immune response.
67
Previous experiments by Young et al (2007) investigated one strain of M. avium WAg206
and showed that animals exposed to these environmental mycobacteria were capable of
suppressing antigen-stimulated IFN-γ production in BCG-immunized mice and of inducing a
de novo IgG antibody response to M. bovis antigens. This is indicative of a Th2 immune
response, which is known to be non-protective. They used the same strain of M. avium that
has been used in this current study.
Therefore the next sets of experiments described in this thesis were carried out to confirm
that the inhibitory effect of environmental mycobacteria on the immune response elicited by
BCG was reproducible. This was necessary before experiments could be performed to
investigate whether a different vaccine strategy could overcome this.
Although tested at a much earlier time-point than Young et al, our results suggested that the
protective Th1 immune response was lowered in BCG vaccinated mice pre-sensitised with M.
avium prior to vaccination. This is supported by the fact that M. avium in particular shares a
large number of similar antigens with BCG, such as Antigen 85 (Ag85) and certain heat
shock proteins (Demangel et al, 2005). In this study, M. avium strain WAg206, a New
Zealand isolate was used. The choice of this strain was based on the experiments carried out
by Young et al. Their findings show that WAg206 has significantly higher inhibitory effect.
WAg206 is an IS901 (Insertion element) positive strain, and this insertion element is shown
to be associated with the virulence (Young et al, 2007). Studies in guinea pigs showed it
affected the protection that BCG offers to live challenge.
The T-cell proliferation assay indicates that the PPD-b specific T-cell response to BCG was
suppressed when the mice were pre-exposed to environmental mycobacteria. Splenocyte
proliferation in response to mycobacterial antigen PPD-b was significantly higher in mice
vacinated with BCG only when compared to mice pre-sensitised to WAg 206. This trend was
68
also observed in T-cell responses to PPD-a. This might be because of the previously
established hypothesis that prior exposure to environmental mycobacteria can prime host’s
immune system against shared mycobacterial antigens which subsequently interferes with the
immune response elicited towards BCG after vaccination, ultimately leading to lowered
protective immune response.
The proliferative responses seen were mimicked in cytokine levels detected. The cytokine
responses were typically Th1 type, and supported the results of the proliferation assay.
PPD-b specific IL-2 was lowered in mice group pre-exposed to WAg206. Interestingly in
response to PPD-avium, the opposite result was seen, as IL-2 production was higher in BCG
vaccinated mice pre-exposed to WAg206 than in BCG only vaccinated mice. This may be
because of the ‘Original antigenic sin’ concept described in 1960 by Thomas Francis, Jr. The
inclination of the body's immune system to preferentially utilize immunological memory
based on a previous infection when a second slightly different version, of that foreign entity
(e.g. a virus or bacterium) is encountered. This leaves the immune system "trapped" by the
first response it has made to each antigen, and unable to elicit potentially more effective
responses during subsequent infections. The phenomenon of original antigenic sin has been
described in relation to influenza virus (Krause & Richard, 2006), dengue fever, human
immunodeficiency virus (HIV), and to several other viruses.
IFN-γ production in response to mycobacterial antigens showed similar response to IL-2. The
IFN-γ production was low in mice pre-exposed to WAg 206 and vaccinated with BCG when
re-stimulated with mycobacterial antigens PPD-b and Ag85. This contrasts with the
response to PPD-avium where IFN-γ production was higher in mice pre-exposed to WAg206
than BCG-only vaccinated mice. These results are similar to those from previous work which
shows that environmental mycobacteria can cross-sensitize the mice to the subsequent IFN-γ
69
response to BCG immunization (Demangel et al., 2005, Brandt et al., 2002). This is because
environmental mycobacteria can prime the host’s immune system against shared
mycobacterial antigens (Brandt et al. 2002). This subsequently interferes with the immune
response elicited towards BCG after vaccination. As IFN-γ is considered as immune mediator
for a Th1 immune response, these lowered IFN-γ levels to PPD-b and Ag85A can be
interpreted as ability of WAg206 to inhibit the vaccine- mediated protection (Young et al.,
2007).
As both IL-2 and IFN-γ were indicative of Th-1 type of immune response, to determine
whether there was evidence of a Th2 response IL-5 production was investigated.
Antigen-specific IL-5 production was very weak (around 20-30 pg/ml) in response to all
antigens suggesting that there was a poor to non-existent Th2 type immune response.
The total antibody levels in serum were no different in both the vaccinated mice groups when
compared to naive (control) mice in response to PPD-b. So, we measured the levels of IgG
subtypes specific to PPD-b and PPD-avium to determine whether there was any difference in
the isotypes which in turn could be indicative of a predominant Th1 or a Th2 response.
Overall, antigen-specific IgG1 isotype levels were higher than IgG2a antibody levels,
confirming previous work (Young et al, 2007). Levels of IgG1 specific to PPD-b were lower
in BCG vaccinated mice pre-exposed to WAg206 than in BCG-only vaccinated mice, and
this trend was reversed with regard to PPD-avium specific IgG1 levels. These results suggest
that antibody levels are directly related to the specific antigen (PPD-b for M. bovis and PPDavium for M. avium) derived from particular mycobacterial species, but the cross-reactivity to
these antigens might induce high level of responses when mice are sensitised to both M.
avium and BCG. This is because both MTB and M. bovis (BCG) are within the genus
Mycobacterium, they share a close relationship at antigen level with environmental
70
mycobacteria as well. This similarity in the nature of antigens shared by them induces a
response with the other antigen which leads to similar but non-protective response.
Based on the results of proliferative and cytokine responses, it was concluded that exposure
to environmental mycobacteria leads to inhibitory effect, and makes BCG vaccine less
immunogenic. This outcome may be influenced by other factors such as dose, route of
immunisation, and interval between pre-sensitisation with M. avium and BCG vaccination.
Though a dose of 106 CFU WAg206 used in this study was generally used in previous studies
by Young et al and others, a more prominent response might be generated by higher doses, or
the use of a more virulent strain (Howard et al., 2002). Brandt et al (2002) used a dose of 2
x106 CFU of environmental mycobacteria to pre-sensitise. They showed no statistically
significant protection against TB when BCG was given. Studies in cattle suggest that
animals that had naturally acquired a higher level of response to M. avium elicited a
comparitively poor response to BCG vaccination and a lower level of immunity compared to
animals with a lower level of naturally acquired response to M. avium tested on previous
occasions (Howard et al., 2002).In addition, the route of exposure might influence the
response: natural oral or respiratory infection may elicit a varied immune response compared
to subcutaneous exposure and have altered outcomes for vaccination (Howard et al., 2002).
4.3 When pre-exposed to environme ntal mycobacte ria, MVA85A proves to be a better
vaccine candidate than VLP/Ag85A
The main challenge now is to create vaccines that over overcome the inhibitory effect created
by environmental mycobacteria, and offers a protective immune response even when the host
is pre-exposed to environmental mycobacteria.
The two main categories of TB candidate vaccines that are being trialled are live
mycobacterial vaccines and subunit vaccines (Reviewed by Anderson & Doherty, 2005).
71
Live vaccines are developed by attenuating virulent organisms, or by genetically modifying
microbes in various ways. This includes adding back deleted genes or increasing the
expression of immunodominant genes. These recombinant vaccines may prime the immune
system more efficiently than BCG, thereby generating a better and more sustained immunity
against TB. One such vaccine is Modified Vaccinia virus Ankara expressing Ag85A
(MVA85A). This vaccine is showing promise as a candidate for BCG because it produces
higher levels of long- lasting cellular immunity when used together with the conventional
BCG (McShane et al., 2004).
Previous work done by Ibanga et al (2006) demonstrated that MVA85A was safe and this
vaccine was shown to induce protective and strong cellular immune responses in humans.
Therefore it was of interest to determine whether the immune respo nse to this live vaccine
was adversely affected after pre-exposure to M.avium WAg206. Responses to this vaccine
were compared with those to VLP/Ag85A, again following pre-exposure to Wag206, because
VLP/Ag85A is an inert vaccine. The use of an effective inert vaccine may avoid public
resistance to the use of genetically modified virus for immunisation.
VLP based vaccines normally induce antibodies and neutralize disease-related proteins, but
they can also induce cell- mediated responses necessary to offer an effective means to treat
mycobacterial diseases (Jennings & Bachmann, 2009).
On the basis of this information the efficacy of two novel vaccines VLP/Ag85A and
MVA85A was compared with BCG following pre-exposure to environmental mycobacteria
M. avium WAg 206.
WAg206 were administered to all groups prior to immunisation and then animals were
vaccinated with different vaccines. MVA85A vaccine proved to be more efficacious than
novel vaccine VLP/Ag85A vaccine with respect to generation of a Th1 immune response.
72
Splenocytes from MVA85A vaccinated mice had higher proliferative response than any other
vaccinated mice in response to all the antigens (PPD-b, PPD-avium, Ag85). VLP/Ag85 did
not induce such high levels of proliferation as seen with MVA85, but was slightly better than
BCG in terms of proliferative response. The response of MVA and VLP vaccinated mice to
PPD a and b may be as a result of M. avium infection (cross reactivity).
Antigen-specific IL-2 responses were unexpectedly different from one antigen to another. In
response to PPD-b, BCG produced slightly higher levels of IL-2 than novel vaccines,
although the levels of IL-2 were low in all groups but, again, in response to PPD-avium and
Ag85, IL-2 production was higher in novel vaccination groups (VLP/Ag85 and MVA85A)
than BCG vaccinated mice. This might be due to specificity of PPD-b to BCG (M. bovis) as
PPD-b antigen is purified protein derivative of M. bovis (BCG). In contrast, the novel vaccine
groups contain the antigen Ag85A so it is likely that they may elicit a better immune response
to Ag85A. These are the reasons most likely to effect to cause variation in cytokine response
among vaccine groups.
Antigen-specific IFN-γ levels were higher in MVA85A mice than any other vaccinated mice,
suggesting a strong Th1 type of immune response on MVA85A vaccination. The point to
note here is IFN-γ levels in response to PPD-b in VLP/Ag85 vaccinated mice was lower than
BCG vaccinated mice, giving a hint that VLP/Ag85 vaccination might not overcome the Th2
type response produced to M. avium.
IL-5 levels specific to Ag85 were much higher in VLP/Ag85 group suggesting the Th2 type
of immune response to VLP/Ag85 vaccination. There were no significant levels of IL-5
produced that were specific to any of the antigens (PPD-b, PPD-avium, Ag85) in either BCG
vaccinated mice or MVA85A vaccinated mice, suggesting that these two groups elicit
protective Th1 immune response.
73
Though there was no significant difference in the levels of serum total antibody levels
specific to PPD-b between different vaccinated mice groups pre-exposed to WAg206 before
vaccination, VLP/Ag85 vaccinated mice produced significantly much higher levels of ser um
total antibody specific to Ag85 than any other group. This further supports that VLP/Ag85
vaccinated mice might have shifted to Th2 type of immune response, which is antibody
mediated.
Overall, IgG1 isotype levels were much higher than IgG2a isotype levels, and VLP/Ag85
vaccinated mice pre-exposed to WAg206 produced significantly higher levels of IgG1
specfic to Ag85 than other groups pre-exposed to WAg206. As IgG1 isotype of antibodies
are indicative of Th2 type of immune response, this result further gives the strength to the
concept that VLP/Ag85 vaccination might have elicited Th2 type of immune response,
instead of protective Th1 immune response. The predominantly Th2 immune response (high
levels of Ag-85 specific IL-5; high level of serum total antibody specific to Ag85; high levels
of IgG1 specific to Ag85) might be because VLP generates higher B-cell mediated immune
response (Bachman et al., 1993). A contributing factor may be that no adjuvant is used in
construction of VLP/Ag85 vaccine. Some adjuvants are known to boost Th1 immune
response and increase the protective immunity (Peacey et al., 2007; Storni et al, 2004; Young
et al., 2006). It may be possible to influence the response to VLP/Ag85 by the selection of
one of these.
Based on results MVA85A vaccine was shown to be a better vaccine candidate than
VLP/Ag85 as it elicited strong Th1 response on vaccination (high proliferative response and
higher IFN-γ production), and this type of cell- mediated immune response is necessary to
develop immunity against TB. This confirms previous work that has established that
MVA85A is an effective vaccine candidate, (McShane et al, 2004).
74
The definitive test of protection against TB is actually challenging the vaccinated animals
with optimal doses of virulent MTB. Trials in which WAg206-treated, MVA/Ag85 or
VLP/Ag85- immunised mice were challenged with live MTB would give further insight into
the value of these vaccines in overcoming the problem of inhibitory environmental
mycobacteria. These trials are underway.
It should be mentioned that the mouse model used in the experiments described in this thesis
has its own disadvantages. Results obtained using this model may not be extrapolated directly
to humans, as the process of granuloma formation in mice after TB infection is different to
that seen in humans and other naturally susceptible hosts (e.g., guinea pigs). Mice are
innately resistant to tuberculosis and the pathogenic effects of MTB do not occur in these
animals unless they are immundeficient. They generate strong protective cellular responses
against TB (Reviewed by Gupta & Katoch, 2009). Despite the differences the mouse model is
the most widely used model of Tb infection. Mice are easily housed and their genome is well
characterised. There are also a wide variety of immunological tools, such as murine-directed
antibodies and gene-deficient strains of mice, which can be utilised when working with
murine models. Subcutaneous BCG vaccination of mice results in Th1 immune response
against mycobacterial antigens. After an aerosol MTB challenge, a ≈1 log10 decrease in lung
bacterial numbers is observed in BCG-vaccinated mice compared to naive controls. This ≈1
log10 decrease is regarded as the gold standard level of protection in pre-clinical vaccine
trials, against which novel vaccines are easily compared. Other animal models such as guinea
pigs that show pathology typical of MTB in humans may provide good vaccine candidates
once the proof of principle is established in mice (Reviewed by Gupta & Katoch, 2009). The
limitations to the use of guinea pigs early in investigations of potential vaccine candidates are
the poor availability of reagents to assess immunologic factors involved in protection in
75
vaccine studies coupled with a limited range of inbred animals enabling repetition of
experiments against a constant genetic background.
4.4 Future directions
Through this research we have been able to conclude that MVA85A is a successful vaccine
candidate that may overcome the inhibitory effect of environmental mycobacteria, by
inducing a strong Th1 type of immune response. However, a VLP based vaccine may also be
successful if an appropriate adjuvant is used in conjunction with this (Jennings and
Bachmann, 2009). Various successful adjuvants being curre ntly used in TB vaccine
preparations listed by Doherty and Andersen (2005) are as follows:

LTK63 (a modified and detoxified heat- labile toxin from E. coli tested in human
volunteers as an influenza vaccine)

Lipovac (a stable emulsion containing the cationic surfactant dimethyl dioctadecyl
ammonium bromide and the synthetic mycobacterial cord factor trehalose dibehenate)

AS2 (an oil- in-water emulsion containing 3-deacylated monophosphoryl lipid A (a
detoxified form of lipid A from Salmonella enterica serovar Minnesota), and a
purified fraction of Quillaria saponaria, known as Quil A)

IC31 (A mixture of oligodeoxynucleotides and polycationic amino acids)

Montanide (A water-oil emulsion, two variants exist, based on mineral and nonmineral oil)
76

ISCOM (A formulation of Quillaja saponins, cholesterol, phospholipids, and protein)

OM-174 (A modified and detoxified lipid A from E. Coli (diphosphate triacyl))
Some of the above mentioned adjuvants might be linked with VLP/Ag85 vaccine in future
studies introducing an immunodrug that offers protective immune response against TB.
Prime-boost immunisation strategies have also been shown to bring about high levels of
cellular immunity against different pathogens. Various studies have demonstrated the
improved effectiveness of these strategies in animal models and the increased
immunogenicity of a BCG prime -MVA85A boost strategy has been established in humans.
Supplementary work on the mode of action of this effective strategy will allow optimisation
of such vaccination strategies, and finally lead to development of an improved vaccine
against TB (McShane and Hill, 2005). So, a combination of priming with MVA85A and
boosting with VLP/Ag85A might lead to better efficacy than a single vaccination as this
proved successful with other novel vaccines. Future work in this area might include strategies
of large scale production of safe and cost-effective VLP based vaccines coupled with
specific mycobacterial antigens that also provide protective immunity against XDR strains of
MTB, that are posing a serious threat.
77
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