Strong Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T-Lymphocyte Activity in Sydney Blood Bank Cohort Patients Infected with nef-Defective HIV Type 1

Proposals for the use of live attenuated human immunodeficiency virus (HIV) type 1 (HIV-1) as a vaccine candidate in humans have been based on the protection afforded by attenuated simian immunodeficiency virus in the macaque model. Although it is not yet known if this strategy could succeed in humans, a study of the Sydney Blood Bank Cohort (SBBC), infected with an attenuated HIV-1 quasispecies with natural nef and nef/long terminal repeat deletions for up to 17 years, could provide insights into the long-term immunological consequences of living with an attenuated HIV-1 infection. In this study, HIV-specific cytoxic T-lymphocyte (CTL) responses in an SBBC donor and six recipients were examined over a 3-year period with enzyme-linked immunospot, tetrameric complex binding, direct CTL lysis, and CTL precursor level techniques. Strong HIV-specific CTL responses were detected in four of seven patients, including one patient with an undetectable viral load. Two of seven patients had weak CTL responses, and in one recipient, no HIV-specific CTLs were detected. High levels of circulating effector and memory HIV-specific CTLs can be maintained for prolonged periods in these patients despite very low viral loads.     

Potent and stable attenuation of live-HIV-1 by gain of a proteolysis-resistant inhibitor of NF-kappaB (IkappaB-alphaS32/36A) and the implications for vaccine development.

Live-attenuated human immunodeficiency viruses (HIVs) are candidates for Acquired Immunodeficiency Syndrome (AIDS) vaccine. Based on the simian immunodeficiency virus (SIV) model for AIDS, loss-of-function (e.g. deletion of accessory genes such as nef) has been forwarded as a primary approach for creating enfeebled, but replication-competent, HIV-1/SIV. Regrettably, recent evidence suggests that loss-of-function alone is not always sufficient to prevent the emergence of virulent mutants. New strategies that attenuate via mechanisms distinct from loss-of-function are needed for enhancing the safety phenotype of viral genome. Here, we propose gain-of-function to be used simultaneously with loss-of-function as a novel approach for attenuating HIV-1. We have constructed an HIV-1 genome carrying the cDNA of a proteolysis-resistant nuclear factor-kappaB inhibitor (IkappaB-alphaS32/36A) in the nef region. HIV-1 expressing IkappaB-alphaS32/36A down-regulates viral expression and is highly attenuated in both Jurkat and peripheral blood mononuclear cells. We provide formal proof that the phenotypic and attenuating characteristics of IkappaB-alphaS32/36A permit its stable maintenance in a live, replicating HIV-1 despite 180 days of forced ex vivo passaging in tissue culture. As compared with other open-reading frames embedded into HIV/SIV genome, this degree of stability is unprecedented. Thus, IkappaB-alphaS32/36A offers proof-of-principle that artifactually gained functions, when used to attenuate the replication of live HIV-1, can be stable. These findings illustrate gain-of-function as a feasible strategy for developing safer live-attenuated HIVs to be tested as candidates for AIDS vaccine.     

New prospects for the development of a vaccine against human immunodeficiency virus type 1. An overview

During the past few years, definite progress has been made in the field of human immunodeficiency virus type 1 (HIV-1) vaccines. Initial attempts using envelope gp120 or gp140 from T-cell line-adapted (TCLA) HIV-1 strains to vaccinate chimpanzees showed that neutralizing antibody-based immune responses were protective against challenge with homologous TCLA virus strains or strains with low replicative capacity, but these neutralizing antibodies remained inactive when tested on primary HIV-1 isolates, casting doubts on the efficacy of gp120-based vaccines in the natural setting. Development of a live attenuated simian immunodeficiency virus (SIV) vaccine was undertaken in the macaque model using whole live SIV bearing multiple deletions in the nef, vpr and vpx genes. This vaccine provided remarkable protective efficacy against wild-type SIV challenge, but the deletion mutants remain pathogenic, notably in neonate monkeys. Study of the mechanisms of protection in the SIV model unravelled the importance of the T-cell responses, whether in the form of cytotoxic T-lymphocyte (CTL) killing activity, or in that of antiviral factor secretion of cytokines, beta-chemokines and other unidentified antiviral factors by CD8+ T-cells. Induction of such a response is being sought at this time using various live recombinant virus vaccines, either poxvirus or alphavirus vectors or DNA vectors, which can be combined together or with a gp120/gp140 boost in various prime-boost combination strategies. New vectors include attenuated vaccinia virus NYVAC, modified vaccinia strain Ankara (MVA), Semliki Forest virus, Venezuelan equine encephalitis virus, and Salmonellas. Recent DNA prime-poxvirus boost combination regimens have generated promising protection results against SIV or SIV/HIV (SHIV) challenge in macaque models. Emphasis is also put on the induction of a mucosal immune response, involving both a secretory IgA response and a mucosal CTL response which could constitute a 'first line of defence' in the vaccinated host. Finally, a totally novel vaccine approach based on the use of Tat or Tat and Rev antigens has been shown to induce efficient protection from challenge with pathogenic SIV or SHIV in vaccinated macaques. The only vaccine in phase 3 clinical trials in human volunteers is a gp120-based vaccine, AIDSVAX. A prime-boost combination of a recombinant canarypoxvirus and a subunit gp120 vaccine is in phase 2. Emphasis has been put recently on the necessity of testing prototype vaccines in developing countries using immunogens derived from local virus strains. Trial sites have thus been identified in Kenya, Uganda, Thailand and South Africa where phase I trials have begun or are expected to start presently.     

Evidence for recombination of live, attenuated immunodeficiency virus vaccine with challenge virus to a more virulent strain

Live, attenuated immunodeficiency virus vaccines, such as nef deletion mutants, are the most effective vaccines tested in the simian immunodeficiency virus (SIV) macaque model. In two independent studies designed to determine the breadth of protection induced by live, attenuated SIV vaccines, we noticed that three of the vaccinated macaques developed higher set point viral load levels than unvaccinated control monkeys. Two of these vaccinated monkeys developed AIDS, while the control monkeys infected in parallel remained asymptomatic. Concomitant with an increase in viral load, a recombinant of the vaccine virus and the challenge virus could be detected. Therefore, the emergence of more-virulent recombinants of live, attenuated immunodeficiency viruses and less-aggressive wild-type viruses seems to be an additional risk of live, attenuated immunodeficiency virus vaccines.     

Novel adeno-associated virus vector vaccine restricts replication of simian immunodeficiency virus in macaques

Gene transfer vectors based on recombinant adeno-associated virus (rAAV) are simple, versatile, and safe. While the conventional applications for rAAV vectors have focused on delivery of therapeutic genes, we have developed the system for delivery of vaccine antigens. In particular, we are interested in generating rAAV vectors for use as a prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine. To that end, we constructed vaccine vectors that expressed genes from the simian immunodeficiency virus (SIV) for evaluation in the monkey SIV model. After a single intramuscular dose, rAAV/SIV vaccines elicited SIV-specific T cells and antibodies in macaques. Furthermore, immunized animals were able to significantly restrict replication of a live, virulent SIV challenge. These data suggest that rAAV vaccine vectors induced biologically relevant immune responses, and thus, warrant continued development as a viable HIV-1 vaccine candidate.     

Genetically engineered live attenuated influenza A virus vaccine candidates.

We have generated new influenza A virus live attenuated vaccine candidates by site-directed mutagenesis and reverse genetics. By mutating specific amino acids in the PB2 polymerase subunit, two temperature-sensitive (ts) attenuated viruses were obtained. Both candidates have 38 degrees C shutoff temperatures in MDCK cells, are attenuated in the respiratory tracts of mice and ferrets, and have very low reactogenicity in ferrets. Infection of mice or ferrets with either mutant conferred significant protection from challenge with the homologous wild-type virus. Three tests for genetic stability were used to assess the propensity for reversion to virulence: 14 days of replication in nude mice, growth at 37 degrees C in tissue culture, and serial passage in ferrets. One candidate, which contains mutations intended to reduce the ability of PB2 to bind to cap structures, was stable in all three assays, whereas the second candidate, which contains mutations found only in other ts strains of influenza virus, lost its ts phenotype in the last two assays. This approach has therefore enabled the creation of live attenuated influenza A virus vaccine candidates suitable for human testing.     

Reduction of simian-human immunodeficiency virus 89.6P viremia in rhesus monkeys by recombinant modified vaccinia virus Ankara vaccination

Since cytotoxic T lymphocytes (CTLs) are critical for controlling human immunodeficiency virus type 1 (HIV-1) replication in infected individuals, candidate HIV-1 vaccines should elicit virus-specific CTL responses. In this report, we study the immune responses elicited in rhesus monkeys by a recombinant poxvirus vaccine and the degree of protection afforded against a pathogenic simian-human immunodeficiency virus SHIV-89.6P challenge. Immunization with recombinant modified vaccinia virus Ankara (MVA) vectors expressing SIVmac239 gag-pol and HIV-1 89.6 env elicited potent Gag-specific CTL responses but no detectable SHIV-specific neutralizing antibody (NAb) responses. Following intravenous SHIV-89.6P challenge, sham-vaccinated monkeys developed low-frequency CTL responses, low-titer NAb responses, rapid loss of CD4+ T lymphocytes, high-setpoint viral RNA levels, and significant clinical disease progression and death in half of the animals by day 168 postchallenge. In contrast, the recombinant MVA-vaccinated monkeys demonstrated high-frequency secondary CTL responses, high-titer secondary SHIV-89.6-specific NAb responses, rapid emergence of SHIV-89.6P-specific NAb responses, partial preservation of CD4+ T lymphocytes, reduced setpoint viral RNA levels, and no evidence of clinical disease or mortality by day 168 postchallenge. There was a statistically significant correlation between levels of vaccine-elicited CTL responses prior to challenge and the control of viremia following challenge. These results demonstrate that immune responses elicited by live recombinant vectors, although unable to provide sterilizing immunity, can control viremia and prevent disease progression following a highly pathogenic AIDS virus challenge.     

A novel self-replicating chimeric lentivirus-like particle

Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4⁺ cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation.     

Involvement of multiple epitope-specific cytotoxic T-lymphocyte responses in vaccine-based control of simian immunodeficiency virus replication in rhesus macaques

Cytotoxic T-lymphocyte (CTL) responses are crucial for the control of immunodeficiency virus replication. Possible involvement of a dominant single epitope-specific CTL in control of viral replication has recently been indicated in preclinical AIDS vaccine trials, but it has remained unclear if multiple epitope-specific CTLs can be involved in the vaccine-based control. Here, by following up five rhesus macaques that showed vaccine-based control of primary replication of a simian immunodeficiency virus, SIVmac239, we present evidence indicating involvement of multiple epitope-specific CTL responses in this control. Three macaques maintained control for more than 2 years without additional mutations in the provirus. However, in the other two that shared a major histocompatibility complex haplotype, viral mutations were accumulated in a similar order, leading to viral evasion from three epitope-specific CTL responses with viral fitness costs. Accumulation of these multiple escape mutations resulted in the reappearance of plasma viremia around week 60 after challenge. Our results implicate multiple epitope-specific CTL responses in control of immunodeficiency virus replication and furthermore suggest that sequential accumulation of multiple CTL escape mutations, if allowed, can result in viral evasion from this control.     

Prevention of disease induced by a partially heterologous AIDS virus in rhesus monkeys by using an adjuvanted multicomponent protein vaccine

Recombinant protein subunit AIDS vaccines have been based predominantly on the virus envelope protein. Such vaccines elicit neutralizing antibody responses that can provide type-specific sterilizing immunity, but in most cases do not confer protection against divergent viruses. In this report we demonstrate that a multiantigen subunit protein vaccine was able to prevent the development of disease induced in rhesus monkeys by a partially heterologous AIDS virus. The vaccine was composed of recombinant human immunodeficiency virus type 1 (HIV-1) gp120, NefTat fusion protein, and simian immunodeficiency virus (SIV) Nef formulated in the clinically tested adjuvant AS02A. Upon challenge of genetically unselected rhesus monkeys with the highly pathogenic and partially heterologous SIV/HIV strain SHIV(89.6p) the vaccine was able to reduce virus load and protect the animals from a decline in CD4-positive cells. Furthermore, vaccination prevented the development of AIDS for more than 2.5 years. The combination of the regulatory proteins Nef and Tat together with the structural protein gp120 was required for vaccine efficacy.     

Studies on virulence of HIV and development of non-virulent live AIDS vaccine using monkeys

A great effort for developing AIDS vaccine has been carried out in the world, designed by various new ideas based on basic research information obtained in recent virology and immunology. Withall it, to obtain effective AIDS vaccine is considered skeptical. One of the reasons of its difficulty is a lack of experimental animals susceptible to HIV-1. In our laboratory, we have succeeded in developing chimeric SIV having 3' half of HIV-1 genome including env (SHIV), which is infectious to macaque monkeys. One of SHIVs has been proved nonpathogenic in monkeys from various aspects and it afforded protective immunity to monkeys against pathogenic SHIV challenge infection. Now, we are trying to develop anti-HIV live attenuated vaccines using the nonpathogenic SHIV as a starting material. In the history of virus vaccine, live attenuated vaccines have been proved most effective in measles and polio-myelitis. However, it is not clear whether nonpathogenic HIV exists or not. Futhermore, even if nonpathogenic HIV could be obtained, there is possibility that it will easily mutate to pathogenic one. Therefore, to develop live attenuated AIDS vaccine is considered dangerous. In this article, We will introduce our research on SHIV pathogenicity using monkeys and hypothesize possibility to obtain nonpathogenic HIV which is speculated from the origin and evolution of HIV/SIV. To clarify virulence and nonvirulence of HIV and to obtain nonpathogenic virus are not only applied research but also basic science to dissolve the fundemental question why HIV can induce the disease.     

A combination DNA and attenuated simian immunodeficiency virus vaccine strategy provides enhanced protection from simian/human immunodeficiency virus-induced disease

Among the most effective vaccine candidates tested in the simian immunodeficiency virus (SIV)/macaque system, live attenuated viruses have been shown to provide the best protection from challenge. To investigate if preimmunization would increase the level of protection afforded by live attenuated SIVmac239Deltanef (Deltanef), macaques were given two priming immunizations of DNA encoding SIV Gag and Pol proteins, with control macaques receiving vector DNA immunizations. In macaques receiving the SIV DNA inoculation, SIV-specific cellular but not humoral responses were readily detectable 2 weeks after the second DNA inoculation. Following boosting with live attenuated virus, control of Deltanef replication was superior in SIV-DNA-primed macaques versus vector-DNA-primed macaques and was correlated with higher levels of CD8+/gamma-interferon-positive and/or interleukin-2-positive cells. Challenge with an intravenous inoculation of simian/human immunodeficiency virus (SHIV) strain SHIV89.6p resulted in infection of all animals. However, macaques receiving SIV DNA as the priming immunizations had statistically lower viral loads than control animals and did not develop signs of disease, whereas three of seven macaques receiving vector DNA showed severe CD4+ T-cell decline, with development of AIDS in one of these animals. No correlation of immune responses to protection from disease could be derived from our analyses. These results demonstrate that addition of a DNA prime to a live attenuated virus provided better protection from disease following challenge than live attenuated virus alone.     

Prior infection with a nonpathogenic chimeric simian-human immunodeficiency virus does not efficiently protect macaques against challenge with simian immunodeficiency virus.

Prior infection with a nef-deleted simian immunodeficiency virus (SIV) protects macaques not only against a homologous pathogenic SIV challenge but also against challenge with a chimeric SIV expressing a human immunodeficiency virus type 1 env gene (SHIV). Since this SHIV is itself nonpathogenic, we sought to explore the use of a nonpathogenic SHIV as a live, attenuated AIDS virus vaccine. Four cynomolgus monkeys infected for greater than 600 days with a chimeric virus composed of SIVmac 239 expressing the human immunodeficiency virus type 1 HXBc2 env, tat, and rev genes were challenged intravenously with 100 animal infectious doses of the J5 clone of SIVmac 32H, an isolate derived by in vivo passage of SIVmac 251. Three of the four monkeys became infected with SIVmac. This observation underlines the difficulty, even with a live virus vaccine, in protecting against an AIDS virus infection.     

Cross-protective immune responses induced in rhesus macaques by immunization with attenuated macrophage-tropic simian immunodeficiency virus.

The simian immunodeficiency virus (SIV) macaque model of AIDS has provided a valuable system with which to investigate vaccine approaches for protection against human immunodeficiency virus type 1 (HIV-1) infection. In particular, the ability of macaques persistently infected with attenuated infectious molecular clones of SIV to resist challenge with the pathogenic parental swarm has conclusively demonstrated that protective immunity can be achieved by immunization prior to exposure. The breadth of these protective responses and the immunological correlates of protection, however, have not been identified. In addition, vaccine studies have mainly employed lymphocyte-tropic strains of HIV-1 and SIV. Recent studies have implicated macrophage-tropic strains in the transmission of HIV-1 and have suggested that these virus strains should be examined in vaccine strategies. Macrophage-tropic viruses may confer additional advantages in the induction of protective immunity by replication in antigen-presenting cells. In this study, the immune response of rhesus macaques inoculated with an attenuated macrophage-tropic recombinant of SIVmac239 (SIV/17E-Cl) was evaluated with respect to protective immunity by heterologous challenge at various times after infection. Vigorous type-specific neutralizing-antibody responses restricted to SIV/17E-Cl were evident by 2 weeks postinfection. By 7 months, however, cross-reactive neutralizing antibodies emerged which neutralized not only SIV/17E-Cl but also the heterologous primary isolate SIV/DeltaB670. Challenge of SIV/17E-Cl-infected monkeys with SIV/DeltaB670 at various times postinfection demonstrated that protective responses were associated with the appearance of cross-reactive neutralizing antibodies. Furthermore, passive transfer of sera from SIV/17E-Cl-infected animals passively protected two of four naive recipients.     

Mucosal AIDS vaccine reduces disease and viral load in gut reservoir and blood after mucosal infection of macaques.

Given the mucosal transmission of HIV-1, we compared whether a mucosal vaccine could induce mucosal cytotoxic T lymphocytes (CTLs) and protect rhesus macaques against mucosal infection with simian/human immunodeficiency virus (SHIV) more effectively than the same vaccine given subcutaneously. Here we show that mucosal CTLs specific for simian immunodeficiency virus can be induced by intrarectal immunization of macaques with a synthetic-peptide vaccine incorporating the LT(R192G) adjuvant. This response correlated with the level of T-helper response. After intrarectal challenge with pathogenic SHIV-Ku2, viral titers were eliminated more completely (to undetectable levels) both in blood and intestine, a major reservoir for virus replication, in intrarectally immunized animals than in subcutaneously immunized or control macaques. Moreover, CD4+ T cells were better preserved. Thus, induction of CTLs in the intestinal mucosa, a key site of virus replication, with a mucosal AIDS vaccine ameliorates infection by SHIV in non-human primates.     

Viral escape from dominant simian immunodeficiency virus epitope-specific cytotoxic T lymphocytes in DNA-vaccinated rhesus monkeys

Virus-specific cytotoxic T lymphocytes (CTL) are critical for control of human immunodeficiency virus type 1 replication. However, viral escape from CTL recognition can undermine this immune control. Here we demonstrate the high frequency and pattern of viral escape from dominant epitope-specific CTL in SIV gag DNA-vaccinated rhesus monkeys following a heterologous simian immunodeficiency virus (SIV) challenge. DNA-vaccinated monkeys exhibited initial effective control of the SIV challenge, but this early control was lost by serial breakthroughs of viral replication over a 3-year follow-up period. Increases in plasma viral RNA correlated temporally with declines of dominant SIV epitope-specific CD8(+) T-lymphocyte responses and the emergence of viral mutations that escaped recognition by dominant epitope-specific CTL. Viral escape from CTL occurred in a total of seven of nine vaccinated and control monkeys, including three animals that initially controlled viral replication to undetectable levels of plasma viral RNA. These data suggest that CTL exert selective pressure on viral replication and that viral escape from CTL may be a limitation of CTL-based AIDS vaccine strategies.     

Dominant induction of vaccine antigen-specific cytotoxic T lymphocyte responses after simian immunodeficiency virus challenge

Cytotoxic T lymphocyte (CTL) responses are crucial for the control of human and simian immunodeficiency virus (HIV and SIV) replication. A promising AIDS vaccine strategy is to induce CTL memory resulting in more effective CTL responses post-viral exposure compared to those in natural HIV infections. We previously developed a CTL-inducing vaccine and showed SIV control in some vaccinated rhesus macaques. These vaccine-based SIV controllers elicited vaccine antigen-specific CTL responses dominantly in the acute phase post-challenge. Here, we examined CTL responses post-challenge in those vaccinated animals that failed to control SIV replication. Unvaccinated rhesus macaques possessing the major histocompatibility complex class I haplotype 90-088-Ij dominantly elicited SIV non-Gag antigen-specific CTL responses after SIV challenge, while those induced with Gag-specific CTL memory by prophylactic vaccination failed to control SIV replication with dominant Gag-specific CTL responses in the acute phase, indicating dominant induction of vaccine antigen-specific CTL responses post-challenge even in non-controllers. Further analysis suggested that prophylactic vaccination results in dominant induction of vaccine antigen-specific CTL responses post-viral exposure but delays SIV non-vaccine antigen-specific CTL responses. These results imply a significant influence of prophylactic vaccination on CTL immunodominance post-viral exposure, providing insights into antigen design in development of a CTL-inducing AIDS vaccine.     

Elicitation of high-frequency cytotoxic T-lymphocyte responses against both dominant and subdominant simian-human immunodeficiency virus epitopes by DNA vaccination of rhesus monkeys

Increasing evidence suggests that the generation of cytotoxic T-lymphocyte (CTL) responses specific for a diversity of viral epitopes will be needed for an effective human immunodeficiency virus type 1 (HIV-1) vaccine. Here, we determine the frequencies of CTL responses specific for the simian immunodeficiency virus Gag p11C and HIV-1 Env p41A epitopes in simian-human immunodeficiency virus (SHIV)-infected and vaccinated rhesus monkeys. The p11C-specific CTL response was high frequency and dominant and the p41A-specific CTL response was low frequency and subdominant in both SHIV-infected monkeys and in monkeys vaccinated with recombinant modified vaccinia virus Ankara vectors expressing these viral antigens. Interestingly, we found that plasmid DNA vaccination led to high-frequency CTL responses specific for both of these epitopes. These data demonstrate that plasmid DNA may be useful in eliciting a broad CTL response against multiple epitopes.