2 курс / Микробиология 1 кафедра / Доп. материалы / Kartikeyan_HIV and AIDS-Basic Elements and Properties
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25.1 – INTRODUCTION
The most important discoveries of the next 50 years are likely to be ones of which, we cannot now even conceive
John Maddox, former editor of Nature
The development of a safe and effective preventive vaccine offers the best hope for containing the HIV epidemic since attempts to modify high-risk behaviour have met with variable results. ARV therapy neither destroys the virus nor cures HIV infection. The objective of ARV treatment is to retard the progress of illness in many patients with advanced disease, and to prevent onset of symptomatic HIV disease in asymptomatic or relatively healthy HIV-positive individuals (Harries et al., 2004; Wig, 2002). A prospective vaccine should limit the initial infecting dose at mucosal surface and produce a rapid systemic cytotoxic T lymphocyte (CTL) response. CTL response can clear a lower viral burden. In such a case, dissemination of HIV to blood stream or organs would not occur (Kent et al., 1997).
HIV infection spreads from a mucosal entry site through regional lymph nodes, to multiple organs. Theoretically, preformed neutralising antibodies, if present in high titres at the site of entry of HIV, may prevent infection of the host. But, the antigenic envelope region of the virus is highly variable. Viruses with variation in the envelope region may escape action of neutralising antibodies (Kent et al., 1997). The replication of HIV to uncontrollably high levels occurs before the formation of anti-HIV immune response. Once HIV replicates to high levels within regional lymph nodes and disseminates throughout the body, the task of eliminating HIV becomes difficult for the following reasons:
1.Soon after infection, HIV replicates in sites, such as the brain, which are not easily accessible to the immune system
2.With ongoing replication, diverse mutants are formed, which escape recognition by CTL (Philips et al., 1991)
3.HIV-infected cells provide a reservoir of HIV, which is difficult to eliminate
4.Immunodeficiency progressively reduces host’s ability to respond to HIV Therefore, HIV should be cleared from the body before it replicates to high levels. A realistic goal of a preventive HIV vaccine would be to reduce viral load (Kent et al., 1997).
25.1.1 – Phases in Vaccine or Drug Trials
In general, new drugs or candidate vaccines are studied in the following phases:
1.Phase zero – preclinical research on pharmacology and toxicology
2.Phase I – randomised controlled clinical trials for safety, pharmacokinetics and pharmacological effects in healthy human volunteers
3.Phase II – randomised controlled double-blind clinical trials for safety and effectiveness in patients
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4.Phase III – efficacy and safety studies in patients with other associated genetic disorders or ailments and in special groups (women of reproductive age, infants, children)
5.Phase IV – extended field trials when the drug or vaccine is in widespread use to study long-term side effects and side effects in special groups
25.2 – STUDY OF IMMUNE RESPONSES
25.2.1 – Immune Responses in those with “Natural Immunity” to HIV
Studies have been conducted in persons who were not infected though they were exposed to HIV perinatally (Rowland-Jones et al., 1993), sexually (RowlandJones et al., 1995), or occupationally (Clerici et al., 1994). Exposed infants and adults, who remain uninfected, have high levels of HIV-specific cytotoxic T-cells (Rowland-Jones et al., 1994; Rowland-Jones et al., 1995). There is also a report of a HIV seropositive baby (born to an HIV-infected mother) who subsequently eliminated HIV (Bryson et al., 1995). The above-mentioned findings indicate that it is possible to clear HIV infection and HIV-specific CD8 CTLs (rather than antibodies) seem to be the major effector mechanism for clearing HIV. While early approaches to develop HIV vaccine concentrated on generating neutralising antibodies, recent approaches have paid attention to generation of HIV-specific CTL responses (Kent et al., 1997).
25.2.2 – Immune Responses in those with “Resistance” to HIV
A mutation in a gene that produces CKR5 (or CCR5 or R5) co-receptor is common among 1 per cent of persons of European descent. CKR5 co-receptor is required for infection by macrophage-tropic strains of HIV. Homozygotes (those possessing one pair of the mutant gene, inherited from both parents) do not have CKR5 co-receptors on their cells and are virtually immune or highly resistant to HIV infection in spite of multiple exposures to the virus (Liu et al., 1996). Thus, macrophage-tropic strains of HIV seem to have a role in initiating infection and these strains could be used as antigens in vaccine preparation (Zhu et al., 1993).
25.2.3 – Study of Local Mucosal Defences
The Peyer’s patches in the gut generate most mucosal activated T- and B-lympho- cytes. These lymphocytes subsequently migrate to other mucosal sites in the gut and genital mucosa. This intermucosal movement of activated lymphocytes constitutes the “common mucosal immune system”. As compared with systemically delivered vaccines, vaccines delivered to any mucosal surface produce more efficient immune response due the common mucosal immune system. Mucosal T-lymphocytes inhibit viral replication (Kent et al., 1997). IgA secreted by relocated B-lymphocytes can prevent absorption of virus into epithelial cells, interfere with assembly of new viruses, and drive IgA–virus complexes back into the lumen (Kent et al., 1997).
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Since there are apprehensions regarding the efficacy of current vaccines based on purified viral proteins and safety of live attenuated vaccines, new approaches are being tried in animal models; however, there is no suitable animal model for HIV-1 infection. These new approaches include:
●Delivering live vector vaccines to mucosal surfaces (e.g. recombinant adenovirus that affects respiratory mucosa; poliovirus, which affects the gut mucosa)
●Incorporating multiple HIV proteins into recombinant live vector or DNA vaccine preparations to broaden anti-HIV immune response
●Injecting purified DNA (that encodes for HIV proteins) into skin or muscle, which produces both antibody and CTL responses
●Incorporating cytokines in vaccine preparations to increase and influence the vaccine-induced immune responses (Kent et al., 1997)
25.2.4 – Role of “Stealth” Liposomes
Liposomes offer multiple advantages as carriers of vaccine antigens as they are biodegradable, non-toxic, synthetic, and elicit both humoral and cell-mediated immunity (CMI). A wide variety of molecules (e.g. drugs, peptides, hormones, enzymes, and genetic materials) could be encapsulated within the aqueous spaces or intercalated into the bilayer membranes or carried on the surface of the liposomes. Manipulations of structural variables (lipid to antigen mass ratio, bilayer fluidity, vesicle size, surface charge, and mode of antigen association with the vesicles) usually induce variation in the level of immune response to a factor of 3. In order to ensure prolonged exposure to immune cells, the liposomes should survive for prolonged periods in vivo. Prolonged survival is achieved by shielding the surface of liposome with polyethylene glycol (PEG) and other hydrophilic polymers, or chemically modifying the hydrophobic part of the phospholipids. This shield fools the phagocytes into ignoring the liposomes. PEGgrafted liposomes are also called “stealth” liposomes. PEG is a synthetic non-toxic polymer. Its molecules get heavily hydrated due to its chemical affinity for water. To phagocytes, this molecular “cloak” of water of hydration makes the PEG-grafted (“stealth”) liposomes appear like watery blobs and tend to ignore them. Consequently, the circulatory life of PEG-grafted liposomes increases and enhances the availability to the immune cells. Stealth liposomes are potentially useful in developing more effective vaccines. PEG-grafted stealth liposomes carrying antigenic epitopes of gp41 (a transmembrane protein of HIV-1) showed about twofold higher immune response and prolonged persistence of antibodies as compared to that of liposomes without PEG moieties (Singh & Bisen, 2006).
25.3 – SCIENTIFIC OBSTACLES
The development of vaccines against HIV poses a formidable challenge. There is inadequate understanding of immunological parameters, which protect against HIV infection or disease, and relationship of genotypic variations to the
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expression of antigens. Some strains of HIV-1 may evade vaccine-induced cellular immunity by genetic diversity, and nullifying anti-HIV cellular immune responses. Occurrence of genotypic mutation and recombination during replication of HIV (Kent et al., 1997). Due to antigenic diversity and hypervariability of HIV, a vaccine protecting against one serotype may not be effective against another subtype or inter-subtype recombinant. Other obstacles to vaccine development include transmission by mucosal route, transmission of HIV by infected cells, resistance of wild-type virus to seroneutralisation, integration of HIV genome into the host cell chromosomes, latency of HIV in resting memory T-cells, rapid emergence of viral escape mutants in the host, and down-regulation of MHC class I antigens (Excler, 2005).
Mucosal Transmission: Since most cases of HIV are due to transmission by the sexual (mucosal) route, efforts are being made to develop a vaccine that would confer protective immunity at the mucosal level. It is necessary to validate the measurement of immune responses at the mucosal level. The CMI-oriented vaccine strategies may not be adapted to the situation since HIV shedding in semen seems to be poorly correlated with systemic CD8 CTL response in humans (Kozlowski & Neutra, 2003).
Animal Models: Lack of appropriate animal models for HIV-1 infection. HIV-1 does not reliably cause AIDS in animal models that can be infected (Kent et al., 1997). In non-human primates, candidate vaccines have elicited immune responses ranging from highly effective to poorly effective in their ability to mitigate infection after challenge with HIV (Desrosiers, 2004). The animal models will be validated when the results in these models are compared with results of phase IIb trials (or “proof of concept” trials) and phase III trials (or “efficacy” trials) in humans (Excler, 2005).
Vaccine Vectors: There is a need to develop vaccine vectors. These vectors “deliver” the genes encoding the target viral immunogens to the antigen-pre- senting cells of the host’s immune system. However, tests conducted on monkeys vaccinated with SIV vaccines have demonstrated SIV-specific CTLs. SIV vaccines seem to delay the onset of AIDS in SIV-infected monkeys (Hirsh et al., 1994). Thus, if a preventive vaccine cannot produce an immune response that can clear HIV infection, it may still achieve the secondary goal of prolonging the dis- ease-free period after HIV infection (Kent et al., 1997).
25.4 – PROGRAMME-RELATED OBSTACLES
The programme-related obstacles in developing an AIDS vaccine include inadequate political leadership, insufficient allocation of funds for AIDS vaccine research, insufficient coordination, lengthy approval process and delays in starting trials in developing countries, and insufficient standardisation of assays and reagents.
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Problems in Recruiting Volunteers: Recruitment of lower-risk volunteers for phase I trials and of at-risk volunteers for phase II and phase III efficacy trials is rendered difficult in less educated populations that are exposed to stigma, discrimination, rumours, misunderstandings, and media opinion (Excler, 2005).
Sample Size: Due to aggressive intervention programmes over the past decade, the incidence rates for HIV infection have shown a decreasing trend, except in some high-risk groups. As a consequence of decreasing incidence rates, vaccine efficacy trials will need to be multicentric or multi-country, in order to achieve the sample size needed at analysis for a given mode of transmission (Excler, 2005).
25.5 – VARIOUS APPROACHES
Classical vaccine strategies based on live attenuated or whole-inactivated HIV has severe limitations (Chertova et al., 2002; Whitney & Ruprecht, 2004). Therefore most efforts to develop AIDS vaccine have focussed on newer vaccine approaches.
25.5.1 – Subunit Vaccine Based on Recombinant Envelope
Soluble glycoprotein of recombinant HIV and SIV have been explored as candidate subunit vaccines. The integrity of envelope glycoprotein seems to be necessary for inducing neutralising antibodies. The current envelope-based candidate vaccines elicit high gp120-binding antibody titres. Antibodies induced by gp120 can neutralise the homologus CXCR4 virus strain but are usually incapable of neutralizing the primary CCR5 isolates (Excler, 2005).
25.5.2 – Subunit Vaccine Based on Tat Protein
Subunit vaccines based on Tat protein have demonstrated partial protective efficacy in animal models and are being tested in humans (Fanales-Belasio et al., 2002).
25.5.3 – Lipopeptides
Lipopeptides have the capability to induce a MHC class I-restricted CD8 response. Long lipopeptides with a fatty acid tail can induce broad cellular immune responses in humans and animals. Lipopeptides with a monolipid tail and a tetanus toxoid peptide appeared to be superior to double lipid tail peptide (Gahery-Segard et al., 2003).
25.5.4 – Live Recombinant Vectors
A live attenuated viral or bacterial strain is used as a vector to carry HIV genes encoding the antigens of interest. Successive injections with the same vector will induce immunity to the vector (Excler, 2005).
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Vaccinia Virus: Though vaccinia virus recombinants were the first vectors to be tried in animals and humans, their possible lack of safety in immune compromised persons has led to the use of attenuated vaccinia virus strains, such as NYVAC HIV-1 subtype C (an attenuated slow-replicative genetically engineered vaccinia virus), and Modified Vaccinia Ankara (MVA) – a highly attenuated, host range-restricted strain of vaccinia virus (Excler, 2005). In non-human primates, MVA recombinants were found to induce potent CTL responses which face pathogenic viral challenge (Im & Hanke, 2004).
Other Pox Viruses: Although canary pox (ALVAC) or fowl pox vectors are very safe in humans (being non-replicative in mammalian cells), they are less immunogenic, as compared to vaccinia virus.
Adenovirus: Recombinants of human adenovirus types 4, 5, and 7 (designated Ad4, Ad5 and Ad7) can be administered orally and intranasally and can induce both systemic and humoral immunity. Adeno-associated viruses (AAVs) are also used.
Alpha Viruses: Defective alpha virus or replicons of Venezuelan equine encephalitis (VEE) virus, Sindbis virus, and Semliki forest virus (SFV) offer the advantages of important amplification of viral message after infection and are able to target dendritic cells (Davis et al., 2002).
Other Viruses: Flavivirus: yellow fever virus; rhabdovirus: vesicular stomatitis virus and rabies virus; myxovirus: influenza virus; paramyxovirus: Sendaï virus, measles virus; picorna virus: polio virus
Bacteria: Bacteria that have been explored as vectors for HIV vaccine include –
Bacillus Calmette Guérin (BCG), Salmonella, Shigella, Lactobacillus, Streptococcus, and Listeria monocytogenes (Fouts et al., 2003).
Limitations of vectors – In individuals previously exposed to the vector and who developed a residual immunity to the vector, most live recombinant viral and bacterial vectors show decreased immunogenicity, as compared to previously unexposed individuals. This limitation is observed with adenovirus type 5 (Ad 5), BCG, poliovirus, MVA and NYVAC (in populations vaccinated against smallpox). However this limitation is not seen with canary pox or fowl pox vectors. In order to overcome the problem of pre-existing immunity to a vaccine vector, efforts now focus on using adenoviruses that rarely affect humans, e.g. Ad11 and Ad35.
25.5.5 – Naked DNA
Intradermal or intramuscular injection of a purified plasmid DNA that carries a gene encoding an antigen usually results in an immune response of Th-1 type (Excler, 2005).
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25.6 – ETHICAL ISSUES
In May 2000, UNAIDS released a guidance document which outlined the strategies to be adopted for capacity building, community involvement, informed consent, protection of vulnerable population, justice and equity in selection of subjects for the clinical trials, use of placebo or any other vaccine in the control arm, continued counselling for risk reduction, inclusion of women and children with adequate safeguards, monitoring mechanism, and care and support for HIV-infection or any associated complication that may occur during the course of the trial (UNAIDS, 2000).
25.6.1 – Continued Counselling for Risk Reduction
There is a possibility that participants in clinical trials of HIV vaccines might abandon safer sexual practices and indulge in risky behaviour due to a false sense of security. Subsequently, if the vaccine is proved to be ineffective, this may lead to increased transmission of HIV. Hence, it is strongly recommended that counselling on safer sexual practices should be carried out along with vaccine trials (Kent et al., 1997; Muthuswamy, 2005). The UNAIDS Document (UNAIDS, 2000) also recommends counselling programme, condom promotion, provision of microbicidal agents for women who are unwilling or cannot use condoms, and control of RTIs and STIs.
25.6.2 – Provision of Antiretroviral Drugs
The vaccine trial protocol should clearly mention that ARV drugs would be provided for those subjects who may become HIV-positive during the course of the vaccine trial. This is in addition to a constellation of services that go beyond what is locally available at the vaccine trial sites (Muthuswamy, 2005).
25.6.3 – Paediatric Trials
Though ARV drugs have shown considerable efficacy in preventing MTCT of HIV, it is not known whether ARV drugs would prevent the transmission of HIV-1 through breast feeding beyond the neonatal period (Gaillard et al., 2004). In addition, infants who escape MTCT are again at risk for HIV infection when they become sexually active as adolescents. Vaccination of infants, begun at birth, is an attractive immunisation strategy. However, development of protective active immunity may take time (Excler, 2005).
25.6.4 – Adolescents
Participation of adolescents in vaccine efficacy trials raises ethical and legal concerns. It is well known that some high-risk groups such as sex workers and MSM, also include adolescents. The growing consensus is that once efficacy is
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demonstrated in adults, bridging studies should be conducted in adolescents (Thorne & Newell, 2004).
25.7 – INDIA’S ROLE IN HIV VACCINE TRIALS
In 2002, it was decided to undertake parallel exploration of several scientific approaches and to embark on parallel implementation of clinical trials so ensure that an effective vaccine is available at the earliest (Excler, 2005).
25.7.1 – Centres of Excellence
Centres of excellence have been set up for clinical and laboratory evaluation of candidate HIV vaccines. This strategy will also help in capacity building for research and development in India. The centres of excellence are National AIDS Research Institute (NARI), Pune where a phase I clinical trial with AAVbased vaccine began in 2005; and Tuberculosis Research Centre, Chennai where a phase I clinical trial with MVA vaccine began in the same year.
25.7.2 – Various Regulatory Mechanisms
Mechanisms have been put in place to ensure transparency and accountability and to address ethical and safety concerns. These include National AIDS Vaccine Advisory Board; Informed Consent Group (to develop a template for informed consent documents to be used in phase I trials); Gender Advisory Board and Training (to incorporate gender concerns in AIDS vaccine trials); NGO Working Group (to ensure that communities are better informed and to obtain community support and representation); and National Consultation on HIV care and treatment (to define policy and technical guidelines for care and treatment of trial participants, including those who may become HIV-positive during the course of the trials).
25.8 – CONCLUSION
The worldwide pursuit for an effective vaccine against AIDS symbolises an unprecedented scientific and human challenge. A preventive vaccine would be the ultimate preventive tool that will complement the existing strategies for prevention. Parallel exploration of several scientific approaches and clinical trials is probably the only way to reach this goal. This unprecedented long-term attempt requires a strong political commitment, flexibility of processes, medical and scientific dedication, and collaboration on a mission mode along with community participation (Excler, 2005).
In 1987, the first candidate HIV vaccine entered clinical trials. Since then, more than 40 candidate vaccines have been evaluated in safety and immunogenicity trials. Between 1987 and 2003, more than 10,000 HIV-negative volunteers have
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participated in these trials. All vaccines tested so far in humans have proven to be safe. One candidate vaccine has progressed to efficacy (phase III) trials (Excler, 2005). Databases have been established by the International AIDS Vaccine Initiative (www.iavi.org) and the United States National Institutes of Health Vaccine Research Center (www.vrc.nih.gov). Several vaccine concepts, immunisation schedules, routes of administration, and adjuvants have been tested (Girard et al., 2004).
In 2005, 13 new trials of preventive candidate AIDS vaccines began in nine countries. India, China, and Russia started their first AIDS vaccine trials in the same year. A randomised, placebo-controlled, dose-escalating, double-blinded trial to evaluate the safety and immunogenicity of TBC-M4 MVA HIV-1 multigenic subtype C vaccine began in December 2005 in India. The antigens used are env, gag, tat-rev, and nef-RT of HIV-1 subtype C. A similar study on tgAACo9 (containing gag, protease and reverse transcriptase of HIV-1 subtype C) has also begun in India. These studies are in phase I. The Aventis-Pasteur live recombinant prime (ALVAC vCP1521) with boost (vax Gen gp120 B/E) is in phase III trial in Thailand (IAVI Report, 2006).
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