HIV-1 Vaccine Development: studies in Macaque models

Since its discovery about 15 years ago, human immunodeficiency virus type 1 (HtV-1) continues·to spread at an alarming rate. It is estimated that by the year 2000 between 30 and 40 million people will be infected with this virus worldwide'. Of these, about 90 % live in developing countries. To date, more than six million people have already developed to the fatal acquired immunodeficiency syndrome (AIDS). Despite numerous research efforts no vaccination or generally accessible therapeutic approach has become available yet. In 1981, the first case of AIDS was recognized'. Epidemiologic studies implicated an infectious agent and in 1983, the isolation of a previously unknown human retrovirus called lymphadenopathy-associated virus (LA V) from an AIDS patient was reported by Barre-Sinoussi et al.'. In 1984, Gallo et aI' and Levy et a/s also reported the isolation of retroviruses from AIDS patients which they called human T-Iymphotropic virus type III (HTLV-III) and AIDS-associated retrovirus (ARV), respectively. Soon thereafter, these retroviruses were recognized as members of the Lentivirinae, a subfamily of the Retroviridae, and were indeed identified as the etiological agents of AIDS. In 1986, these retroviruses were named human immunodeficiency virus (HIV). Today, two types of HIV are recognized, HIV-1 and HIV-2. Of these, HIV-1 is the primary etiologic agent of the current AIDS pandemic. The search for an HIV-1 counterpart in other primate species led to the identification of several simian immunodeficiency viruses (SIV), which may cause AIDS like syndromes in monkeys'. Currently, macaque lentivirus infections are considered the most appropriate animal models to study HIV pathogenesis and possible intervention strategies. In the following section, a concise overview is given of the biology of primate lentiviruses, including the host immune response and correlates of immune-mediated protection. Aspects important for vaccine development are highlighted

Chemical inactivation of recombinant vaccinia viruses and the effects on antigenicity and immunogenicity of recombinant simian immunodeficiency virus envelope glycoproteins.

The efficiency of paraformaldehyde (PFA) and binary ethylenimine (BEI) in inactivating recombinant vaccinia virus (rVV), present in baby hamster kidney cells expressing simian immunodeficiency virus envelope glycoproteins (SIV-Env), was measured in a series of inactivation studies. Both compounds were shown to be effective in reducing rVV titres. The use of standard 3-day titration assays proved to be inadequate to measure PFA inactivation, since upon prolonged incubation, residual rVV infectivity was detected in cultures negative at 3 days. Different procedures using PFA or BEI were selected to assess their influence on the antigenicity and immunogenicity or rVV expressed SIV-Env. Antigenicity, as defined by the ability to react with a panel of monoclonal antibodies recognizing major antigenic sites, and immunogenicity, as defined by the ability to induce SIV envelope specific and virus neutralizing serum antibodies in rats, proved to be preserved after either inactivation procedure. These data show that both protocols using

Simian immunodeficiency virus (SIV)-specific CD8+ cytotoxic T lymphocyte responses of naive and vaccinated cynomolgus macaques infected with (SIV)mac32H(J5): quantitative analysis by in vitro antigenic stimulation.

Detailed analyses of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocyte (CTL) responses in vaccinated and infected macaques may help to clarify the role of CTL immunity in protection against lentiviruses. Here, the optimal conditions for the measurement of SIV Gag-specific CTL were investigated by bulk and limiting dilution assays of peripheral blood mononuclear cells (PBMC) from naive and vaccinated cynomolgus macaques (Macaca fascicularis) infected with SIVmac32H(J5). In vitro restimulation was generally required for CTL detection. Selective activation of CD8+ and MHC-restricted SIV Gag-specific CTL was induced by stimulation with autologous para-formaldehyde-fixed B-lymphoblastoid cell lines infected with a recombinant vaccinia virus expressing SIV Gag. Applied to limiting dilution assays, antigenic stimulation reproducibly demonstrated SIV Gag-specific CTL precursors (CTLp) in PBMC of all animals studied, including those lacking significant responses in standard bulk CTL assays

Antigenicity and immunogenicity of recombinant envelope glycoproteins of SIVmac32H with different in vivo passage histories.

Shortly after infection of two rhesus monkeys (Macaca mulatta) either with a SIVmac32H challenge stock or with the same virus that had been passaged in another rhesus monkey for 11 months, SIV-envelope genes were cloned from their peripheral blood mononuclear cells and subsequently expressed by recombinant vaccinia viruses. The molecular weights and antigenicities of the thus produced envelope glycoproteins were largely identical to those of the native SIV. The envelope glycoprotein derived from the in vivo passaged virus proved to be poorly recognized by virus neutralizing monoclonal antibodies directed against one of the seven antigenic sites for which monoclonal antibodies were available. Immunization studies in rats showed that this protein was also less efficient in inducing antibodies against this antigenic site, and that it induced significantly lower levels of virus neutralizing antibodies than the other SIV-envelope glycoprotein. The immunogenicity of the SIV-envelope glycoprotein incorporated into immune stimulating complexes (iscoms) was compared to that of the same protein presented with Quil A or MDP-tsl

Vaccine-induced virus-neutralizing antibodies and cytotoxic T cells do not protect macaques from experimental infection with simian immunodeficiency virus SIVmac32H (J5).

To gain further insight into the ability of subunit vaccines to protect monkeys from experimental infection with simian immunodeficiency virus (SIV), two groups of cynomolgus macaques were immunized with either recombinant SIVmac32H-derived envelope glycoproteins (Env) incorporated into immune-stimulating complexes (iscoms) (group A) or with these SIV Env iscoms in combination with p27gag iscoms and three Nef lipopeptides (group B). Four monkeys immunized with recombinant feline immunodeficiency virus Env iscoms served as controls (group C). Animals were immunized intramuscularly at weeks 0, 4, 10, and 16. Two weeks after the last immunization, monkeys were challenged intravenously with 50 monkey 50% infectious doses of virus derived from the J5 molecular clone of SIVmac32H propagated in monkey peripheral blood mononuclear cells. High titers of SIV-neutralizing antibodies were induced in the monkeys of groups A and B. In addition, p27gag-specific antibodies were detected in the monkeys of group B. Vaccine-induced cytotoxic-T-lymphocyte precursors against Env, Gag, and Nef were detected on the day of challenge in the monkeys of group B. Env-specific cytotoxic-T-lymphocyte precursors were detected in one monkey from group A. In spite of the observed antibody and T-cell responses, none of the monkeys was protected from experimental infection. In addition, longitudinal determination of cell-associated virus loads at weeks 2, 4, 6, 9, and 12 postchallenge revealed no significant differences between vaccinated and control monkeys. These findings illustrate the need to clarify the roles of the different arms of the immune system in conferring protection against primate lentivirus infections

Decline of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocytes in the peripheral blood of long-term nonprogressing macaques infected with SIVmac32H-J5.

The evolution of simian immunodeficiency virus (SIV)–specific cytotoxic T lymphocyte precursors (CTLps) and their relationship with virus replication were studied in SIV‐infected macaques. After primary viremia, 3 of 8 macaques l

CD8+ cytotoxic T lymphocytes of a cynomolgus macaque infected with simian immunodeficiency virus (SIV)mac32H-J5 recognize a nine amino acid epitope in SIV Gag p26.

A detailed analysis of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocyte (CTL) responses and the identification of the proteins and epitopes they target may improve the design of immunotherapeutic interventions and provide insights into AIDS pathogenesis. Here, we identified a new CTL epitope in the SIV Gag protein, recognized by CD8+ and MHC class I-restricted CTL clones from a long-term asymptomatic cynomolgus macaque (Macaca fascicularis) infected with SIVmac32H-J5. Using overlapping synthetic peptides, the optimal minimal epitope was characterized as a nine amino acid peptide representing amino acids 242-250 of p26 (SVDEQIQWM). CTL recognition was shown to be abolished by amino acid substitutions observed within homologous human immunodeficiency virus (HIV)-1 and HIV-2 sequences