Rodjana Chunhabundit
HIV Vaccine Production
Introduction
In addition to the production of protease inhibitor, our group also thinks of the
alternative ways to fight the AIDS pandemic. One of these is the production of an HIV
vaccine. HIV vaccine is necessary for Thailand that has high infection rate from many high
risk groups such as commercial sex workers, intravenous drug users and sexual transmitted
disease patients, and the budget for the high cost drug therapies is not available. At the
present time, Thailand participates in HIV vaccine clinical trials. Thus, our production group
will evaluate the HIV vaccine by focusing mainly on the techniques for vaccine production
and less on the process of clinical trials and regulatory approval of HIV vaccine. Additional
information based on the answers of the questions that were raised after my presentation will
also be addressed in this report.
The Need for HIV Vaccine
By the year 2000, it is estimated that 50 million individuals will be infected with HIV
and, by 2010, the death toll could climb to 40 million. Even with today’s advances in
research and treatment, diagnosis of HIV infection in most parts of the world is still a death
sentence. In developing countries where the HIV infection rate has been soaring, the cost of
palliative care of HIV-infected individuals and AIDS patients far exceeds the total amount
the governments of those countries are able to spend on their nation’s general health care [1].
Even in wealthy, industrialized nations that antiviral therapeutics are available, poor
toleration of the drugs and the emergence of resistant viruses make long-term responses to
antiviral therapies far from universal. Moreover, replication-competent HIV-1 persists in
lymphoid tissue even in individuals who appear to have responded well to antiviral therapy.
The development and distribution of a safe, effective and inexpensive preventive HIV
vaccine currently represents the best hope for controlling the global HIV/AIDS pandemic.
Requirements for An HIV Vaccine
Vaccination protects a recipient from pathogenic agents by establishing an
immunological resistance to infection. The requirements for an HIV vaccine follow the same
principles of immunology applied to other successful vaccines. A successful vaccine must
induce all of the following:
1. Neutralizing antibody to prevent infection with cell-free virus.
2. Lymphokines to limit the spread of infection.
3. Cell mediated immunity (CMI), especially cytotoxic T cells (CTLs) to ensure
recovery from infection.
4. Long-lasting memory T and B cells to ensure sustained protection [2].
Current Strategies for an HIV-1 Vaccine
There are now 5 technologies for making vaccines.
1. Inactivated or killed vaccines
2. Live attenuated vaccines
3. Subunit vaccines
- Recombinant envelope protein
- Peptide vaccine
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4. Live vector-based vaccines
5. DNA vaccines (DNA plasmids)
Inactivated or Killed Vaccines.
Inactivated or killed vaccines are made from whole viruses that are killed or
inactivated by some chemicals. The strongest argument for a whole killed HIV virus is that
antigens are presented in a fashion similar to the way they are presented by the real pathogen,
therefore the immune system can respond effectively. However, inactivated whole viruses
are considered impractical for AIDS because of the fears that incomplete inactivation of HIV
viruses might lead to inadvertent infection of vaccine [4].
Preparation of the inactivated whole-virus vaccine derived from a proviral DNA clone of SIV
1. Preparation of the infectious SIV
1.1 Transfecting a proviral DNA clone of SIV from sooty mangbeys into CEMx 174 cells
1.2 Supernatants from expanded cultures are harvested, clarified, and concentrated 100
fold by ultrafiltration.
1.3 Concentrated virus is further purified by ultracentrifugation through a 20% glycerol
cushion
1.4 Pelleted, partially purified virus is resuspended in phosphate-buffered saline and store
at –70oC until inactivation.
2. Inactivation of virus
2.1 Add 10 l of 5 mg/ml solution of psoralen (trioxsalen) to 5 ml of concentrated virus
at room temperature.
2.2 Expose the suspension of virus to UV light (365 nm at a distance of 5 cm) for 15 min.
2.3 Repeat psoralen/UV light treatment 3 times.
2.4 Dilute inactivated virus in buffer, aliquot and store in liquid nitrogen.
2.5 Inactivation is confirmed by inoculating CEMx 174 cells in culture with the vaccine
preparation [5].
Another method of virus inactivation is incubation at 4oC for 24 hrs with 0.8% formalin [6].
Live Attenuated Vaccines
Genetic manipulation may be used to construct modified organisms (bacteria or
viruses) that are used as live recombinant vaccines. These vaccines are either nonpathogenic
organisms that have been engineered to carry and express antigenic determinants from a
target pathogenic agent or engineered strains of pathogenic organisms in which the virulent
genes have been modified or deleted. In these instances, as a part of a bacterium or virus, the
important antigenic determinants are presented to the immune system with a conformation
that is very similar to the form of the antigen in the disease-causing organism. Live
attenuated vaccine strains provide a sustained stimulus to the humoral and cellular immune
systems. By contrast, purified antigen alone often lacks the native conformation and elicits a
weak immunogenic response [7].
Several companies are seriously considering developing live attenuated HIV as a
vaccine candidate because infection with an attenuated strain of HIV is believed to be the
reason why some HIV-infected people are long-term non-progressors. A vaccine candidate
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based on a mutant strain of HIV that infected 9 Australians 17 years ago has been developed
in Australia. The strain of virus that was transmitted to these 9 individuals lacks a large
segment of HIV’s nef gene. This apparently limits the damage of the immune system from
HIV virus [8]. However, a live attenuated virus vaccine is generally considered impractical
for AIDS because of the possibilities of reversion of an attenuated strain. Moreover, insertion
of viral cDNA from a vaccine strain may enhance expression of cellular oncogenes leading to
malignant transformation caused by the promoters in the HIV genome.
The Limitations of Traditional Vaccines
- Production of animal and human viruses requires animal cell culture, which is
expensive.
- Both the yield and rate of production of animal and human viruses in culture are
often quite low, thereby making vaccine production costly.
- Extensive safety precautions are necessary to ensure that laboratory and production
personel are not exposed to a pathogenic agent.
- Insufficiency killing or attenuation during the production process can introduce
virulent organisms into the vaccine and inadvertently spread the disease.
- Attenuated strains may revert, a possibility that requires continual testing to ensure
the reacquisition of virulence has not occurred.
- Not all diseases (e.g., AIDS) are preventable through the use of traditional vaccines.
Therefore, recombinant DNA technology has provided a means of creating a new
generation of vaccines that overcome the drawbacks of traditional vaccines [7].
Subunit Vaccines
Vaccines that use components of a pathogenic organism rather than the whole
organism are called “subunit” vaccines; recombinant DNA technology is very well suited for
developing new subunit vaccines. There are advantages and disadvantages to the use of
subunit vaccines. On the positive side, using purified protein(s) as an immunogen ensures
that the preparation is stable and safe, is precisely defined chemically, and is free of
extraneous proteins and nucleic acids that can initiate undesirable side effects in the host
organism. On the negative side, purification of a specific protein can be costly and, in certain
instances, an isolated protein may not have the same conformation as it does in situ, with the
result that its antigenicity is altered. Another serious shortcoming of HIV subunit vaccines
relates to the genetic variation among individual isolates of HIV. This variability is most
extensive in the env gene. In pre-clinical evaluation of candidate envelope gp vaccines, the
antibody response of immunized animals are often type specific, recognizing only the
envelope of the immunized virus strain. HIV-2 clearly shows only 50-60% genetic homology
with HIV-1 and its envelope gp is antigenically distinct. Thus, successful vaccination against
one virus would not necessarily confer immunity against other viruses. Multivalent vaccines
containing different envelope glycoproteins from strains of both HIV-1 and HIV-2 may be
required to confer immunity against the divergent strains present in nature.
Large-scale production of a vaccinia recombinant derived HIV-1 gp 160
Based on other retroviral models, such as murine, feline, bovine and simian viruses,
the obvious candidate for a vaccine to prevent primary infection is the envelope glycoprotein.
The major envelope glycoprotein gp120 contains sites for binding to the receptor CD4, and it
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also contains sites that are recognized by neutralizing antibodies. There have also been
reports that T-cell mediated immune responses can be generated by immunization with
gp120 or a recombinant vaccinia virus coding for the HIV gp160. It has been reported that
neutralizing epitopes are present on both the gp120 and gp41 proteins and injection of
chimpanzees with purified gp120 alone has failed to provide protection against infection with
HIV-1. For these reasons, a candidate vaccine consisting of the full-length envelope
glycoprotein has been developed.
HIV-1 envelope glycoproteins can be purified from virus or infected cells but only in
small amounts which make large-scale vaccine studies difficult. Therefore, alternative
approaches are cloning and expression of envelope glycoproteins in bacterial, mammalian
and insect cell systems. However systems that are not mammalian systems result in
production of proteins which are nonglycosylated or have different glycosylation patterns.
This could result in alterations in the folding of the protein and the loss of conformation
epitopes important in the induction of a specific humoral and cellular immune response.
The following procedures describe a gp160 expression system based on coinfection
of Vero cells with two recombinant vaccinia viruses which is a useful vector for the envelope
glycoprotein gene. When properly engineered the gp proteins are synthesized, processed and
transported to the membrane of infected cells. Although vaccinia virus leads to cell death,
there is little lysis and the majority of cells remain intact, allowing easy extraction of the
required protein from infected cells.
Materials and methods.
1. Construction of plasmids
- plasmid containing the gp160 gene flanked by bacteriophage T7 promotor and
termination sequence
-plasmid containing T7 RNA polymerase gene under the regulation of the vaccinia
7.5 promotor
2. Construction of vaccinia virus recombinants.
Infecting CV-1 cells with vaccinia virus and transfecting them with calcium
phosphate precipitated plasmid DNA. The cells are harvested, and TK-recombinant
viruses are isolated.
3. Production of recombinant virus stocks
High titer stocks of vaccinia recombinants are prepared by infecting Vero cells with
1 pfu virus/cell. After 2-3 days incubation at 37oC, the infected cells are shaken into the
medium and pelleted by centrifugation at 5000 g for 20 min. The supernatant is poured
off and stored at 4oC. The cells are then washed three times in PBS. A trypsin solution is
then added to the cell suspension to give an end concentration of 0.025 % trypsin. After
incubation at 37oC for 30 min, the trypsinized cell suspension is then pooled with the
medium supernatant and this is aliquoted and frozen at –80oC. This procedure increases
the virus titer to approximately 10-fold of that presented in cell medium alone
4. Large-scale cultivation of Vero cells
A Vero cell inoculum is first prepared by passage of cells in Roller bottles (Nunc) to
produce sufficient cells to inoculate a 6 liter fermenter, and then used as inoculum for a
40 liter vessel. During cultivation in 6 liter and 40 liter fermenter, the cells are allowed
to adhere to microcarriers.
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5. gp160 Production
When a cell density of 5x109/liter is achieved the microcarriers are settled and the
medium is pumped out. Then the recombinant viruses are pumped into the fermenter. After
virus adsorption the fermenter is filled with medium, Over a 40 hr period, the fermenter is
perfused with 40 liters of the same medium. At this time about 80% of the cells are detached
from microcarrier and are pumped out with the medium. The remaining adhered cells are
detached by washing and rapid stirring. Microcarriers are then removed by passage of this
suspension through a 70 m sieve. The cell containing gp160 are then pelleted by
centrifugation and stored at –80oC before processing for gp160 extraction and purification.
6. Purification
- Cell pellet is thawed, resuspended, and passed through a homogenizer.
- The cell membrane resuspension is pelleted by centrifugation and resuspended.
- The suspension is then clarified by centrifugation.
- The supernatant is then incubated with lentil-lectin sepharose that is used for
absorbtion of glycoproteins
- Bound proteins are eluted and then incubated with sepharase–bound monoclonal
antibody to HIV gp160.
- After washing, and elution, the elutate is dialysed against TBS.
- The dialyzed eluate is adjusted to 1% Zwittergent and 5% Betain and adsorbed to a
MonoQ matrix.
- This is then eluted and detergents are removed by a second lentil-lectin
chromatography step.
- The glycoprotein is eluted and dialyzed again [9].
Peptide Vaccines
These vaccines are derived from target epitopes that are most immunogenic or
peptide sequences that bind neutralizing antibody most strongly, for example, synthetic
amino acid sequence 735 to 752 of gp160, synthetic peptide RP-135 that is a 24 amino acid
fragment of gp120 or synthesized segments of p17, a core protein conserved among different
HIV-1 strains.
Even though they are highly specific, relatively inexpensive and safe, a disadvantage
of synthetic peptides over peptides purified from culture or produced from cloned genes is
that tertiary structure may be different from that of the natural protein and may not stimulate
antibodies that react with the natural protein. Epitopes that are not linearly continuous, but
are brought together by folding of the native peptide, may be critical determinants.
Preparation of the hybrid T1-SP10 carrier-free peptide
T1 = HIV env gp120 T-cell epitope
SP10 = B cell epitopes from V3 loop region.
To induce anti-HIV CTL responses as well as neutralizing antibody responses.
Materials and methods
1. Peptides are synthesized on an Applied Biosystems 431A peptide synthesizer using tertbutoxycarbonyl chemistry
2. Synthesized peptides are deprotected and cleaved from the supporting resin with
hydrogen fluoride in 10% Anisole
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3. Peptides are solubilized in 15-25% (vol/vol) glacial acetic acid and lyophilized.
4. Peptides are reconstituted in endotoxin-free phosphate-buffered saline (PBS) and
dialyzed or are HPLC purified and the molecular mass determined by mass spectrometry
[10].
Live Vector-Based Vaccine
This is the use of recombinant techniques to clone HIV-1 genes of interest into live
virus vectors such as vaccinia virus, avian pox virus or adenovirus. Such a recombinant
vaccine has the advantage of allowing sustained expression of large amount of HIV-1 antigen
and, thereby, stimulation of neutralizing antibody and cellular immune responses.
Vaccinia virus is a member of the pox virus family. Vaccinia virus DNA replicates
in the cytoplasm of infected cells, rather than in the nucleus, because vaccinia virus DNA
contains genes for DNA polymerase, RNA polymerase and the enzymes to cap, methylate
and polyadenylate mRNA.Thus, if a foreign gene is inserted into the vaccinia virus genome
under the control of a vaccinia virus promoter, it will be expressed independently of host
regulatory and enzymatic functions. Because the vaccinia virus has a broad host range, is
well characterized at the molecular level, is stable for years after lyophilization, and is
usually benign virus, it is a strong candidate as a vector vaccine. However, the vaccinia virus
genome is very large and lacks unique restriction sites, so it is not possible to insert
additional DNA directly into the viral genome. Of necessity, the genes for specific antigens
must be introduced into the viral genome by in vivo homologous recombination.
Method for the integration of a gene into vaccinia virus
1. The DNA sequence coding for a specific antigen is inserted into a plasmid vector
immediately downstream of a cloned vaccinia virus promotor and in the middle of a
nonessential vaccinia virus gene such as the gene for the enzyme thymidine kinase
2. The plasmid is used to transfect chick embryo fibroblasts in culture, that have previously
been infected with wild-type vaccinia virus, which produces a functional thymidine
kinase
3. Recombination between DNA sequences that flank the promotor and the antigen gene on
the plasmid and the homologous sequences on the viral genome results in the
incorporation of the cloned gene into the viral DNA.
4. The absence of the thymidine kinase gene in the host cells and the disruption of the
thymidine kinase gene in the recombined virus render the host all resistant to the toxic
effects of bromodeoxyuridine. This character is used as selection system for cell lines that
carry a recombinant vaccinia virus.
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Fig. 1 Integration of cloned antigen gene into vaccinia virus
DNA Vaccines
DNA immunization is a novel approach for the elicitation of protective immune
response because it involves the in vivo delivery of antigen-encoding plasmid DNA rather
than actual protein immunogens. The de novo production of correctly folded and
glycosylated antigens in vivo following DNA administration effectively elicits both humoral
and cytotoxic cellular immune responses, and can confer protection against live virus
challenge. A cloned gene encoding an antigen on a plasmid is used to coat the gold particles.
Delivery of DNA vaccines into epidermis is done by using a particle acceleration or “gene
gun” device that employs a controlled electric discharge to create a shock wave that
accelerates DNA-coated gold particles into a given target tissue. By using the gene gun the
number of plasmid vector copies delivered per cell and the depth of tissue penetration can be
controlled. However, simple intramuscular injection can also be used to introduce the DNA
vaccine. Comparisons between direct DNA inoculation and gene gun-based DNA delivery
indicate that substantially stronger immune responses can be obtained with two or three
orders of magnitude less DNA when a gene gun based delivery is used [11].
Regulatory Requirements for Clinical Development of Candidate Vaccines
After development, to qualify for licence in United States, vaccines must meet the
standards and requirements stated in the Code of Federal Regulations (Title 21, part 600) for
safety, purity, potency, immunogenicity and efficacy. Pre-clinical tests must be conducted in
vitro, in small animals, and sometimes in nonhuman primates to assess the safety, toxicity,
and immunogenicity of a candidate vaccine. After the pre-clinical tests have been completed
successfully, the sponsor (e.g., investigator, university, commercial firm, and government
agency) can submit a Notice of Claimed Exemption for a New Drug to the U.S. FDA. The
investigated vaccine is then referred to as an investigated new drug (IND).
The IND application must include description of the composition, source, and
manufacturing process of the vaccine; quality control and the methods used to test the
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vaccine’s safety, purity, and potency; and a summary of all laboratory and pre-clinical animal
tests. Moreover, a protocol detailing the clinical study and consent form approved by the
local institutional review board and the names and qualifications of the clinical investigators
should be included. During a 30-day period, the IND application is reviewed by the FDA to
determine whether human subjects will be exposed to unwarranted risks. Vaccine lots for
clinical trials must be produced according to standard good manufacturing practices. If the
vaccine is safe and efficacious in clinical trials, an application for a product license
application (PLA) can be submitted to the FDA. FDA approval for licence is based on a
satisfactory review of all data in the PLA, the results of confirmatory tests by the FDA on
product samples from manufactures, and an inspection of the production facilities [12].
Clinical Trials of HIV Vaccines
Although useful information is frequently obtained by studies in animals, animal
models can only approximate the pathophysiology of, and immune response to, disease in
humans. Therefore, the determination of the effect of a vaccine in humans must ultimately
rest on the actual administration of the vaccine to humans.
Design of HIV vaccine trials
Clinical trials of viral vaccines are carried out in a series of three phases.
Phase I trials primarily address the issue of safety. Since HIV antigens may possess
immunoregulatory activities, clinical evaluation of experimental HIV vaccine will require a
more comprehensive immunoanalysis than other viral vaccines. Thereby, the sample size and
the length of follow up is greater than in that phase I studies of other viral vaccines. Finally,
phase I trials address preliminary immunogenicity questions with the aim of identifying safe
and immunogenic doses which can be further evaluated in later phase of vaccine testing.
Population for phase I HIV vaccine trials should be individuals at no risk for HIV infection.
Phase II trials will involve larger numbers of volunteers and will be designed to
determine optimal dosage schedules based on safety and immunogenicity data. Phase II trials
will be studied in both high and low risk populations, in order to determine whether the
vaccine is safe and immunogenic in persons at high risk and compare responses among the
various population groups.
Phase III trials will be carried out as placebo-controlled, randomized, and double
blind studies. The number of volunteers in each group determined by incidence of infection
rates in the population being studied and statistical parameters sought for analysis of
protection from infection. Because of large numbers of volunteers and difficulties to
determine the efficacy of HIV vaccines, it is anticipated that only the most promising vaccine
candidates will be evaluated in phase III. The several population groups at high risk for HIV
infection could be used in phase III efficacy studies [9].
Potential target populations for Phase III HIV vaccine trials
- Homosexual men
- Intravenous drug users
- Commercial Sex Workers
- Military personnel in regions of high HIV incidence
- Newborns of seropositive mothers
- Sexual transmitted disease patients
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Vaccines under Clinical Investigation
Since 1988, the U.S. National Institutes of Allergy and Infection Diseases (NIAID)
has supported a network of several academic institutions and corporations, referred to as the
AIDS Vaccine Evaluation Group (AVEG), to evaluate the safety and immunogenicity of
candidate HIV vaccines in phase I and II clinical trials [12].
AIDS Vaccine Evaluation Group (AVEG) Vaccine Trials [13]
# Subunit vaccines (Phase I, II)
- Recombinant envelope protein: gp160 IIIB, gp160 MN, gp120 IIIB, gp120 MN,
and gp120 SF2
- Peptide vaccines: V3 peptide, T-B peptide
# Live vector-based vaccines (Phase I, II)
- vaccinia-env
- vaccinia-env/gag/pol
- canarypox-env
- canarypox-env/gag mucosal
The University of Pensylvania collaborated with Apollon, Inc. of Malvern [14]
# DNA Vaccine (phase I)
- Plasmid containing env and rev genes of HIV MN
VaxGen, Inc. [1]
In 1997, VaxGen’s team developed two “bivalent” AIDSVAX formulations,
designed to protect against the major strains of HIV. The formulation that would be used in
clinical trials in North America was designed to protect against the strains of the virus
typically found in America, Western Europe, and Australia. The formulation to be used in
Thailand trials was designed to protect against the strains typical in Thailand, Korea, Japan,
Indonesia, and Taiwan. In early 1998, VaxGen began to test bivalent forms of AIDSVAX in
Phase I/II trials. These studies followed earlier trials of monovalent AIDSVAX. More than
1,200 persons have been inoculated with monovalent and bivalent AIDSVAX through
clinical trials in the U.S. and Thailand. The vaccines induced antibodies in more than 99% of
inoculated individuals. Now, VaxGen has taken the vaccine into Phase III clinical trials to
study the efficacy of AIDSVAX in preventing infection by HIV. These clinical trials are the
first large-scale efficacy trials for an HIV vaccine in the world.
Summary of VaxGen’s Phase III Trials of AIDSVAX
Location
Vaccine
Sites
Volunteers
Number
Start
Length
Endpoints
North America
AIDSVAX B/B
61
High-risk heterosexuals
5,400
June, 1998
3 yrs
Infection & viral load
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Thailand
AIDSVAX B/E
17
Injection drug users
2,500
March, 1999
3 yrs
Infection & viral load
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HIV Vaccine Clinical Trials in Thailand
HIV vaccine clinical trial should be performed in Thailand for several reasons. For
example :
- Thailand still has high infection rate of HIV, the number of individuals infected with
HIV will be 1.3 million by the year 2000.
- Thai government reveals political policy in the prevention and control AIDS.
- Thailand has experience in other vaccine developments such as hepatitis B virus and
encephalitis virus.
- HIV infection endemic in Thailand is caused by HIV subtype B and E. Subtype E is
the strain typical of the AIDS causing virus in Thailand, Japan, Korea, Taiwan and Indonesia.
- An HIV vaccine that consists of rgp120 of subtype B and subtype E has been
developed already. Therefore the clinical trial of this vaccine in Thailand can determine the
efficacy of the vaccine for protection of high risk individuals from acquiring infection.
The chronology of HIV vaccine clinical trials in Thailand
- The first study performed by Thai Red Cross in 1994 was the Phase I trial of an
HIV-1 synthetic peptide vaccine produced by UBI company.
- The Phase I/II trial of MN rgp120 HIV-1 vaccine (Genentech, Inc.) among
intravenous drug users in Bangkok is conducted by the cooperation between Bangkok
Metropolitan Administration (BMA), Vaccine Trial Center of Faculty of Tropical Medicine
and Faculty of Medicine Siriraj Hospital; Mahidol University.
- The phase I/II of HIV rgp120 SF2/MF59 vaccine trials were performed by the
Armed Forces Research Institute of Medical Sciences (AFRIMS) and Health Science
Institute of Chiang Mai University in 1995.
- The Phase I of Immunogen vaccine trial was studied in 30 infected individuals in
1996 by Faculty of Medicine Ramathibodi Hospital; Mahidol University.
- The cooperated studies between AFRIMS, Health Science Institute of Chiang Mai
University, Vaccine Trial Center of Faculty of Tropical Medicine and Faculty of Medicine
Siriraj Hospital; Mahidol University are conducting the Phase I/II rgp120 subtype E vaccine
trials for the first time in the world [15].
- The Vaccine Trial Center of Faculty of Tropical Medicine, Mahidol University
begined the Phase I/II trials of AIDSVAX B/E and Thai E vaccine in 1997 and in
cooperation with the BMA and ministry of Public Health, the efficacy test of AIDSVAX B/E
in 1998 [16].
The Results from Pre-clinical and Clinical Trials of rgp 160
Pre-clinical tests
The study illustrated the protection of macaques against SIV infection by subunit
vaccines of SIV envelope glycoprotein gp160 was conducted by Hu et al. Four macaques
(Macaca fascicularis) were first immunized with recombinant vaccinia virus expressing
SIVmne gp160. All animals seroconverted with low-titer antibodies to SIVmne gp120 and
gp32. However, immunized animals also showed activated SIV-specific helper T-cell
function. At week 62 and 70, all four immunized macaques were boosted intramuscularly
with subunit gp160 produced in baculovirus-infected insect cells in order to enhance specific
B-cell response to SIV envelope antigens. Within 2 weeks of the first gp160 boost, all
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animals showed a dramatic (30 to 50 fold) increase in antibody response against SIV
envelope antigens. This increase was also concordant with a significant rise in serum
neutralizing activities against both SIVmne and SIVmac 251. The antibody titers declined 5 to
10 fold during the next 8 weeks but seemed to stabilize after the second gp160 boost at week
70. At week 74, the four immunized animals, together with four control macaques were
challenged with an intravenous inoculation of the homologous virus SIVmne. After the
challenge, the four control animals seroconverted within 4 to 7 weeks and virus in cocultures
of lymphocytes were detected at 2 to 4 weeks after infection. In contrast, all four immunized
remained virus-negative (longer than 1 year after the challenge) based on virus isolation and
polymerase chain reaction analysis. No significant infection was observed in four animals
that were inoculated intravenously with lymph node cells and peripheral blood mononuclear
cells (PBMC) collected from each of the four immunized animals at 46 weeks after
challenge. Thus, the results indicated that a “sterilizing immunity” against the challenge
infection was achieved in the immunized animals [17].
Clinical trials
As an immunotherapy in HIV-infected individuals:
- In uncontrolled trials, the gp160 vaccine appeared to broaden binding antibody
response and boost cellular immune responses as measured by lymphoproliferative
responses, delayed-type hypersensitivity reactions and CD4+/CD8+ CTL activities. The
immune responses were more pronounced in those who received frequent booster
immunizations and in those with higher CD4 levels.
- In a controlled phase II trial conducted in Sweden by Wahren et al., six doses of
gp160 induced strong T-cell responses against a variety of HIV antigens. Most of the
vaccinated individuals had improved CD4 counts during the first year of the trial, and some
had increases in neutralizing antibodies and antibody-dependent cellular cytotoxicity [12].
- In U.S., a phase II efficacy trial was conducted with rgp160 in 608 HIV-infected,
asymptomatic volunteers with CD4+ cell counts >400 cells/mm3. During a 5-year study
volunteers received a 6-shot primary series of immunizations with either rgp160 or placebo
over 6 months followed by booster every 2 months. Adequate follow-up and acquisition of
endpoints allowed for definitive interpretation of the trial results. There was no evidence that
rgp160 has efficacy as a therapeutic vaccine in early-stage HIV infection [18].
Future Trend of HIV Vaccine
The National Institute of Health (NIH) decided not to fund large-scale efficacy trials
of the leading HIV vaccines since 1994. Now NIH plans to speed up vaccine development by
several steps, including boosting funding; creating a new peer-review study section to
evaluate vaccine proposals; launching a set of standardized, comparative tests of candidate
AIDS vaccines in monkeys and trying to stimulate partnerships between U.S. investigators
and colleagues in other countries. These changes come at a time when NIH has been under
heavy criticism for not doing enough to speed the search for an AIDS vaccines. NIH’s AIDS
research budget in 1999 spent nearly $180 million on vaccine researches, a 79% jump from
1995. This increased funding has begun to lure researchers into the field. NIH also increased
emphasis on monkey studies in order to move human versions of the most promising vaccine
forward. Moreover, NIH takes part in tests of VaxGen’s gp120 that is one of the vaccine it
rejected in 1994 [19].
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The biggest demand for an HIVvaccine will be in developing countries so it is
unlikely to produce substantial profits for manufacturers. The new incentives to draw
companies into public/private partnerships will help to increase political commitment to the
national AIDS vaccine programs that are now starting up in countries such as China, South
Africa, Russia, and Brazil. Another way is to stimulate support from industrialized nations
for international efforts, such as that led by the World Bank and a joint initiation between
UNAIDS and the World Health Organization [20].
Conclusion
Traditional vaccine designs for viral diseases are largely based on attenuated viruses
or, killed viruses. But such approaches are unlikely to be directly applicable to HIV.
Alternative vaccine design and development efforts that take unique properties of HIV
pathogenesis and epidemiology into consideration will be employed to create a new
generation of vaccines that provide effective prevention for the targeted population.
The preventive HIV vaccine should have the following criteria.
Elicition of specific CTL and neutralizing antibody for all strains of HIV
Ease of administration
Safe
Low cost
However, when a new vaccine is developed, it must go through many stages of
scientific testing before it is approved for widespread use. In case of AIDS, preventive or
post–exposure or therapeutic vaccines are still needed. The efficiency of vaccines to elicit the
HIV specific CTL and neutralizing antibody responses that mediate this protection can be
increased through the application of currently available technologies. Finally, increased
collaborations, private interest, and investment will eventually speed the discovery of a
successful HIV vaccine.
References
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4. Woodrow GC and Levine MM. New Generation Vaccines. New York: Marcel Dekker,
Inc. 1990.
5. Johnson PR, Montefiori DC, Goldstein S, et al. Inactivated whole-virus vaccine derived
from a proviral DNA clone of simian immunodeficiency virus induces high levels of
neutralizing antibodies and confers protection against heterologous challenge. Proc. Natl.
Acad. Sci. USA 1992; 89: 2175-2179.
6. Corb MM, Martin LN, Fairburn BD, et al. A formalin-inactivated whole SIV vaccine
confers protection in macaques. Science 1989; 255: 1293-1297.
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