Showing papers by "David C. Montefiori published in 1999"

Control of Viremia in Simian Immunodeficiency Virus Infection by CD8+ Lymphocytes

Abstract: Clinical evidence suggests that cellular immunity is involved in controlling human immunodeficiency virus-1 (HIV-1) replication. An animal model of acquired immune deficiency syndrome (AIDS), the simian immunodeficiency virus (SIV)-infected rhesus monkey, was used to show that virus replication is not controlled in monkeys depleted of CD8+ lymphocytes during primary SIV infection. Eliminating CD8+ lymphocytes from monkeys during chronic SIV infection resulted in a rapid and marked increase in viremia that was again suppressed coincident with the reappearance of SIV-specific CD8+ T cells. These results confirm the importance of cell-mediated immunity in controlling HIV-1 infection and support the exploration of vaccination approaches for preventing infection that will elicit these immune responses.

Neutralizing antibody-independent containment of immunodeficiency virus challenges by DNA priming and recombinant pox virus booster immunizations.

Abstract: Eight different protocols were compared for their ability to raise protection against immunodeficiency virus challenges in rhesus macaques. The most promising containment of challenge infections was achieved by intradermal DNA priming followed by recombinant fowl pox virus booster immunizations. This containment did not require neutralizing antibody and was active for a series of challenges ending with a highly virulent virus with a primary isolate envelope heterologous to the immunizing strain.

Protection by Live, Attenuated Simian Immunodeficiency Virus against Heterologous Challenge

Abstract: We examined the ability of a live, attenuated deletion mutant of simian immunodeficiency virus (SIV), SIVmac239Delta3, which is missing nef and vpr genes, to protect against challenge by heterologous strains SHIV89.6p and SIVsmE660. SHIV89.6p is a pathogenic, recombinant SIV in which the envelope gene has been replaced by a human immunodeficiency virus type 1 envelope gene; other structural genes of SHIV89.6p are derived from SIVmac239. SIVsmE660 is an uncloned, pathogenic, independent isolate from the same primate lentivirus subgrouping as SIVmac but with natural sequence variation in all structural genes. The challenge with SHIV89.6p was performed by the intravenous route 37 months after the time of vaccination. By the criteria of CD4(+) cell counts and disease, strong protection against the SHIV89.6p challenge was observed in four of four vaccinated monkeys despite the complete mismatch of env sequences. However, SHIV89.6p infection was established in all four previously vaccinated monkeys and three of the four developed fluctuating viral loads between 300 and 10,000 RNA copy equivalents per ml of plasma 30 to 72 weeks postchallenge. When other vaccinated monkeys were challenged with SIVsmE660 at 28 months after the time of vaccination, SIV loads were lower than those observed in unvaccinated controls but the level of protection was less than what was observed against SHIV89.6p in these experiments and considerably less than the level of protection against SIVmac251 observed in previous experiments. These results demonstrate a variable level of vaccine protection by live, attenuated SIVmac239Delta3 against heterologous virus challenge and suggest that even live, attenuated vaccine approaches for AIDS will face significant hurdles in providing protection against the natural variation present in field strains of virus. The results further suggest that factors other than anti-Env immune responses can be principally responsible for the vaccine protection by live, attenuated SIV.

Highly attenuated vaccine strains of simian immunodeficiency virus protect against vaginal challenge: inverse relationship of degree of protection with level of attenuation.

Abstract: Three different deletion mutants of simian immunodeficiency virus (SIV) that vary in their levels of attenuation were tested for the ability to protect against mucosal challenge with pathogenic SIV. Four female rhesus monkeys were vaccinated by intravenous inoculation with SIVmac239Delta3, four with SIVmac239Delta3X, and four with SIVmac239Delta4. These three vaccine strains exhibit increasing levels of attenuation: Delta3 < Delta3X

A Canarypox Vaccine Expressing Multiple Human Immunodeficiency Virus Type 1 Genes Given Alone or with Rgp120 Elicits Broad and Durable CD8+ Cytotoxic T Lymphocyte Responses in Seronegative Volunteers

Abstract: Induction of CD8 + cytotoxic T cells is considered one of the important correlates for the protective efficacy of candidate human immunodeficiency virus type 1 (HIV-1) vaccines. To induce CD8 + cytotoxic T lymphocytes (CTLs) along with neutralizing antibody and CD4 + T cell help, a live canarypox virus construct expressing gp120, transmembrane gp41, the gag and protease genes, and sequences containing CTL epitopes in nef and pol was given simultaneously with, or followed by, rgp120 SF2. CD8 + CTLs were detected in 61% of volunteers at some time during the trial. Three to 6 months after the last immunization, the gene-specific responses were gag, 26/81; env, 17/77; nef, 12/77; and pol, 3/16. Simultaneous immunization with the canarypox vector and the subunit, beginning with the initial immunization, resulted in earlier antibody responses. In summary, a strategy of immunization with a canarypox vector expressing multiple genes of HIV-1 given with gp120 results in durable CD8 + CTL responses to a broad range of epitopes.

Lack of Viral Escape and Defective In Vivo Activation of Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Lymphocytes in Rapidly Progressive Infection

Abstract: Human immunodeficiency virus type 1 (HIV-1)-specific immune responses over the course of rapidly progressive infection are not well defined. Detailed longitudinal analyses of neutralizing antibodies, lymphocyte proliferation, in vivo-activated and memory cytotoxic T-lymphocyte (CTL) responses, and viral sequence variation were performed on a patient who presented with acute HIV-1 infection, developed an AIDS-defining illness 13 months later, and died 45 months after presentation. Neutralizing-antibody responses remained weak throughout, and no HIV-1-specific lymphocyte proliferative responses were seen even early in the disease course. Strong in vivo-activated CTL directed against Env and Pol epitopes were present at the time of the initial drop in viremia but were quickly lost. Memory CTL against Env and Pol epitopes were detected throughout the course of infection; however, these CTL were not activated in vivo. Despite an initially narrow CTL response, new epitopes were not targeted as the disease progressed. Viral sequencing showed the emergence of variants within the two targeted CTL epitopes; however, viral variants within the immunodominant Env epitope were well recognized by CTL, and there was no evidence of viral escape from immune system detection within this epitope. These data demonstrate a narrowly directed, static CTL response in a patient with rapidly progressive disease. We also show that disease progression can occur in the presence of persistent memory CTL recognition of autologous epitopes and in the absence of detectable escape from CTL responses, consistent with an in vivo defect in activation of CTL.

Characterization of Primary Isolate-Like Variants of Simian-Human Immunodeficiency Virus

Abstract: Several different strains of simian-human immunodeficiency virus (SHIV) that contain the envelope glycoproteins of either T-cell-line-adapted (TCLA) strains or primary isolates of human immunodeficiency virus type 1 (HIV-1) are now available. One of the advantages of these chimeric viruses is their application to studies of HIV-1-specific neutralizing antibodies in preclinical AIDS vaccine studies in nonhuman primates. In this regard, an important consideration is the spectrum of antigenic properties exhibited by the different envelope glycoproteins used for SHIV construction. The antigenic properties of six SHIV variants were characterized here in neutralization assays with recombinant soluble CD4 (rsCD4), monoclonal antibodies, and serum samples from SHIV-infected macaques and HIV-1-infected individuals. Neutralization of SHIV variants HXBc2, KU2, 89.6, and 89.6P by autologous and heterologous sera from SHIV-infected macaques was restricted to an extent that these viruses may be considered heterologous to one another in their major neutralization determinants. Little or no variation was seen in the neutralization determinants on SHIV variants 89.6P, 89.6PD, and SHIV-KB9. Neutralization of SHIV HXBc2 by sera from HXBc2-infected macaques could be blocked with autologous V3-loop peptide; this was less true in the case of SHIV 89.6 and sera from SHIV 89.6-infected macaques. The poorly immunogenic but highly conserved epitope for monoclonal antibody IgG1b12 was a target for neutralization on SHIV variants HXBc2, KU2, and 89.6 but not on 89.6P and KB9. The 2G12 epitope was a target for neutralization on all five SHIV variants. SHIV variants KU2, 89.6, 89.6P, 89.6PD, and KB9 exhibited antigenic properties characteristic of primary isolates by being relatively insensitive to neutralization in peripheral blood mononuclear cells with serum samples from HIV-1-infected individuals and 12-fold to 38-fold less sensitive to inhibition with recombinant soluble CD4 than TCLA strains of HIV-1. The utility of nonhuman primate models in AIDS vaccine development is strengthened by the availability of SHIV variants that are heterologous in their neutralization determinants and exhibit antigenic properties shared with primary isolates.

Neutralization escape in human immunodeficiency virus type 1-infected long-term nonprogressors.

Abstract: Neutralization-escape variants of human immunodeficiency virus type 1 (HIV-1) were sought in persons who had persistent low virus loads and who remained asymptomatic for at least 12-16 years of infection without antiretroviral therapy. Viruses were isolated from 3 persons at two or three time points during the course of infection and were assessed for neutralization by sequential autologous serum samples. Virus neutralization was poor or undetectable with contemporaneous autologous serum but improved with later serum samples for each person. In particular, later isolates resisted neutralization by autologous serum samples that neutralized an earlier isolate. Strain-specific neutralizing antibodies remained detectable for up to 4.2 years without diminishing in titer. The results demonstrate that neutralization-escape variants arise periodically in HIV-1-infected long-term nonprogressors.

Role of Immune Responses against the Envelope and the Core Antigens of Simian Immunodeficiency Virus SIVmne in Protection against Homologous Cloned and Uncloned Virus Challenge in Macaques

Abstract: We previously showed that envelope (gp160)-based vaccines, used in a live recombinant virus priming and subunit protein boosting regimen, protected macaques against intravenous and intrarectal challenges with the homologous simian immunodeficiency virus SIVmne clone E11S. However, the breadth of protection appears to be limited, since the vaccines were only partially effective against intravenous challenge by the uncloned SIVmne. To examine factors that could affect the breadth and the efficacy of this immunization approach, we studied (i) the effect of priming by recombinant vaccinia virus; (ii) the role of surface antigen gp130; and (iii) the role of core antigens (Gag and Pol) in eliciting protective immunity. Results indicate that (i) priming with recombinant vaccinia virus was more effective than subunit antigen in eliciting protective responses; (ii) while both gp130 and gp160 elicited similar levels of SIV-specific antibodies, gp130 was not as effective as gp160 in protection, indicating a possible role for the transmembrane protein in presenting functionally important epitopes; and (iii) although animals immunized with core antigens failed to generate any neutralizing antibody and were infected upon challenge, their virus load was 50- to 100-fold lower than that of the controls, suggesting the importance of cellular immunity or other core-specific immune responses in controlling acute infection. Complete protection against intravenous infection by the pathogenic uncloned SIVmne was achieved by immunization with both the envelope and the core antigens. These results indicate that immune responses to both antigens may contribute to protection and thus argue for the inclusion of multiple antigens in recombinant vaccine designs.

Toward an HIV type 1 vaccine that generates potent, broadly cross-reactive neutralizing antibodies.

Abstract: P ROGRESS IN T HE D EV EL OPM E NT of an effective vaccine for HIV-1 has been gauged in large part by the ability to induce measurable virus-specific CD8 1 cytotoxic T lymphocytes (CTLs) and neutralizing antibodies as lead correlates of a protective immune response. 1±3 Although there has been success in generating low-level CTLs in a subset of vaccinated volunteers in phase I clinical trials, little progress has been made in generating neutralizing antibodies. Part of the problem for neutralizing antibodies is the high degree of genetic variability in the HIV-1 surface gp120 and transm embrane gp41 glycoproteins. 7 Although conserved neutralization epitopes exist, the current repertoire of candidate HIV-1 vaccines fails to generate sufficient antibodies to them. Critical features of the structure of the oligomeric envelope glycoproteins as they exist in their native form on the virus present yet another level of complexity for immunogen design. Most candidate HIV-1 envelope subunit vaccines are based on monomeric gp120 from T cell line-adapted (TCLA) strains of virus. Those vaccines have generated neutralizing antibodies with variable and sometimes potent activity against the vaccine strain, low activity against heterologous TCLA strains, and poor activity against primary isolates. It is conceivable that this narrow specificity, particularly the inability to neutralize primary isolates, will greatly limit the effectiveness of the antibody component of a vaccine. Scientists have been divided on the issue of whether the weak neutralizing antibody response generated by candidate HIV-1 vaccines is a property of the vaccine or the assay. A major argument has been that current assays underestimate neutralization potency, an argument that stems mostly from the fact that primary isolates are generally less sensitive to neutralization than TCLA strains. A concentrated effort has been made to resolve the vaccine-versus-assay issue by focusing mainly on the assay. We now recognize that primary isolates are relatively insensitive to neutralization under a variety of conditions, 27±30 including those that take into account the differential use of CXCR4 and CCR5 as coreceptors. 31±33 These developments, combined with features revealed by the crystal structure of gp120 and gp41, are making it increasingly clear that the dichotomy in neutralization sensitivity between TCLA strains and primary isolates is unrelated to the assay and actually reflects fundamental differences in the structure of the oligomeric envelope glycoproteins of these two categories of virus. Thus, repeated passage of HIV-1 in T cell lines has the consequence of selecting virus variants on which the neutralization epitopes are readily accessible for efficient antibody binding. Some of these epitopes reside in the V3 cysteine±cysteine loop of gp120, which is a major target for neutralizing antibodies on TCLA strains but not primary isolates. The Division of AIDS, Preventive and Vaccine Branch of the National Institutes of Health (Bethesda, MD), convened a workshop in October of 1998 to discuss new strategies to improve the neutralizing antibody responses generated by candidate HIV-1 vaccines. The goals of the workshop were four-fold: (1) consider the advantages and disadvantages of using genetically engineered cell lines to assess a neutralizing antibody response, (2) examine evidence from passive immunization experiments showing that potent neutralization is required for protection, (3) review the structure of gp120 and gp41 revealed by X-ray crystallography as it relates to neutralization and (4) discuss the merits of novel immunization strategies in early phases of development. A summary of this workshop is presented here.

Limited Breadth of the Protective Immunity Elicited by Simian Immunodeficiency Virus SIVmne gp160 Vaccines in a Combination Immunization Regimen

Abstract: We previously reported that immunization with recombinant simian immunodeficiency virus SIVmne envelope (gp160) vaccines protected macaques against an intravenous challenge by the cloned homologous virus, E11S. In this study, we confirmed this observation and found that the vaccines were effective not only against virus grown on human T-cell lines but also against virus grown on macaque peripheral blood mononuclear cells (PBMC). The breadth of protection, however, was limited. In three experiments, 3 of 10 animals challenged with the parental uncloned SIVmne were completely protected. Of the remaining animals, three were transiently virus positive and four were persistently positive after challenge, as were 10 nonimmunized control animals. Protection was not correlated with levels of serum-neutralizing antibodies against the homologous SIVmne or a related virus, SIVmac251. To gain further insight into the protective mechanism, we analyzed nucleotide sequences in the envelope region of the uncloned challenge virus and compared them with those present in the PBMC of infected animals. The majority (85%) of the uncloned challenge virus was homologous to the molecular clone from which the vaccines were made (E11S type). The remaining 15% contained conserved changes in the V1 region (variant types). Control animals infected with this uncloned virus had different proportions of the two genotypes, whereas three of four immunized but persistently infected animals had >99% of the variant types early after infection. These results indicate that the protective immunity elicited by recombinant gp160 vaccines is restricted primarily to the homologous virus and suggest the possibility that immune responses directed to the V1 region of the envelope protein play a role in protection.

Protection of Macaques against Intrarectal Infection by a Combination Immunization Regimen with Recombinant Simian Immunodeficiency Virus SIVmne gp160 Vaccines

Abstract: We previously reported that immunization with recombinant simian immunodeficiency virus SIVmne envelope (gp160) vaccines protected macaques against intravenous challenge by the cloned homologous virus E11S but that this protection was only partially effective against the uncloned virus, SIVmne. In the present study, we examine the protective efficacy of this immunization regimen against infection by a mucosal route. We found that the same gp160-based vaccines were highly effective against intrarectal infection not only with the E11S clone but also with the uncloned SIVmne. Protection against mucosal infection is therefore achievable by parenteral immunization with recombinant envelope vaccines. Protection appears to correlate with high levels of SIV-specific antibodies and, in animals protected against the uncloned virus, the presence of serum-neutralizing activities. To understand the basis for the differential efficacies against the uncloned virus by the intravenous versus the intrarectal routes, we examined viral sequences recovered from the peripheral blood mononuclear cells of animals early after infection by both routes. We previously showed that the majority (85%) of the uncloned SIVmne challenge stock contained V1 sequences homologous to the molecular clone from which the vaccines were made (E11S type), with the remainder (15%) containing multiple conserved changes (the variant types). In contrast to intravenously infected animals, from which either E11S-type or the variant type V1 sequences could be recovered in significant proportions, animals infected intrarectally had predominantly E11S-type sequences. Preferential transmission or amplification of the E11S-type viruses may therefore account in part for the enhanced efficacy of the recombinant gp160 vaccines against the uncloned virus challenge by the intrarectal route compared with the intravenous route.

Postexposure immunoprophylaxis of primary isolates by an antibody to HIV receptor complex.

Abstract: mAb B4 is a monoclonal antibody directed against HIV receptor complex. The antibody had broad neutralizing activity against HIV and provided postexposure prophylaxis to hu–peripheral blood leukocyte (PBL)–severe combined immunodeficient mice and chimpanzees. B4 recognized a complex receptor site for HIV on the T cell surface that includes CD4 and also may be influenced by interaction with HIV coreceptors. mAb B4 preferentially neutralized primary HIV-1 isolates compared with T cell line-adapted strains, including syncytium-inducing and non-syncytium-inducing phenotypes, representatives from HIV-1 subtypes A-G, as well as HIV-2, simian immunodeficiency virus, and chimeric simian/human immunodeficiency virus (SHIV). Neutralization was demonstrated in both pre- and postinfection models. The administration of mAb B4 after infectious challenge totally interrupted the infection of hu-PBL–severe combined immunodeficient mice by PBL-grown HIV-1 and the infection of chimpanzees by chimp-adapted HIV-1. This mode of protection suggested that the anti-HIV receptor antibody is efficacious for prophylaxis after exposure to HIV and for prevention of maternal transmission and may be an effective antiretroviral agent for treatment.

Primary Isolate Neutralization by HIV Type 1-Infected Patient Sera in the Era of Highly Active Antiretroviral Therapy

Abstract: Sera from highly selected HIV-1-positive patients are known to have the ability to neutralize a diverse array of primary isolates of HIV-1. The human osteosarcoma cell line that expresses CD4 and chemokine receptors (GHOST cells) was adapted to study HIV-1 neutralization in 37 HIV-1-infected individuals who were selected because of slow disease progression or nonprogression. Many of these individuals were receiving combination drug therapy. Molecularly cloned HIV-1 JR-FL and NL4-3 viruses were used as prototypes to define assay conditions. Sera were then tested at a 1:40 dilution against six additional primary isolates, three of which utilized CCR5 and three of which used both CCR5 and CXCR4. The assay was highly reproducible and independent of viral input titer, with a readout at 48 hr equivalent to that at later time points. As previously reported, neutralization sensitivity was entirely independent of coreceptor usage. Only a few sera from slow progressors were able to neutralize a broad array of primary isolates at a 1:40 dilution, and the best clinical predictor of broadly neutralizing antibody for primary isolates was the present use of antiretroviral agents. In further studies it was found that purified antibody accounted for the majority of the measured neutralization. However, experiments with exogenous addition of antiviral agents showed that the use of nucleosides also greatly contributed to the measured neutralization in some patients. Measurement of neutralization of HIV-1 primary isolates by sera from patients receiving antiretroviral therapy must be carried out with some caution.

Magic of the Occult

Abstract: The AIDS epidemic continues unabated, undaunted by researchers' attempts to develop a vaccine for this disease. Now a new, promising method for raising potentially effective antibodies to HIV is reported in this week's issue ([ LaCasse et al ][1].). In their Perspective, Montefiori and Moore describe the ambitious experiments that led to this discovery and the work that needs to be done before it is clear whether these results will pave the way to stemming the spread of AIDS. [1]: http://www.sciencemag.org/cgi/content/short/283/5400/357

Neutralizing antibody responses in Africa green monkeys naturally infected with simian immunodeficiency virus (SIVagm).

Abstract: This study assessed the magnitude and cross-reactivity of the neutralizing antibody response generated by natural SIV infection in wild-caught African green monkeys. Neutralizing antibodies of variable potency, sometimes exceeding a titer of 1:1,000, were detected in 20 of 20 SIV-seropositive African green monkeys in Kenya. Detection of those neutralizing antibodies was dependent on the strain of virus and the cells used for assay, where the most sensitive detection was made with SIVagm1532 in Sup T1 cells. Potent neutralization of SIVagm1532 was seen with contemporaneous autologous serum. Potent neutraliza- tion was also detected with laboratory-passaged SIVmac25 1 and SIVsmB670, but not with SIVsmE660 and two additional strains of SIVagm. Serum samples from rhesus macaques (Macaca mulatta) ex- perimentally infected with either SIVmac25 1 or SIVsmE660 were capa- ble of low-level neutralization of SIVagm. These results indicate that natural infection with SIV can generate strain-specific neutralizing anti- bodies in African green monkeys. They also indicate that some neutral- ization determinants of SIVagm are partially shared with SIV strains that arose in sooty mangabys and were subsequently transmitted to 1 rhesus macaques.

Combined systemic and mucosal immunization with microsphere-encapsulated inactivated simian immunodeficiency virus elicits serum, vaginal, and tracheal antibody responses in female rhesus macaques.

Abstract: We determined the efficacy of immunization with microsphere-encapsulated whole inactivated simian immunodeficiency virus (SIV) by combined systemic and mucosal administration to protect female rhesus macaques against vaginal challenge with homologous rhesus PBMC-grown SIVmac251. Animals in one group were primed and boosted intramuscularly. Two groups were primed intramuscularly and boosted either intratracheally or orally. A final group was primed by vaccinia/rgp140 scarification and subdivided for either intratracheal or oral boosting. Strong ELISA titers of circulating SIV-specific IgG and modest IgA responses were elicited in the animals primed intramuscularly. Intratracheal boosting in the intramuscularly primed macaques resulted in high bronchial alveolar wash (BAW) IgG and less pronounced IgA. SIV-specific vaginal wash (VW) IgG was also present in the intramuscular/intramuscular and intramuscular/intratracheal groups. Vaccinia/rgp140 priming gave low ELISA titers to whole SIV, and failed to elicit m...

Immunization against SIVmne in macaques using multigenic DNA vaccines.

Abstract: All structural and regulatory genes of SIVmne were cloned into mammalian expression vectors to optimize expression in vitro and immunogenicity in mice. Macaca fascicularis were immunized four times with plasmid DNA (n = 4), or two DNA priming inoculations followed by two boosts of recombinant gp160 plus Gag-Pol particles (n = 4). Following intrarectal challenge with SIVmne, all macaques became infected. Three monkeys immunized with DNA alone maintained low plasma virus loads by 1 year post-challenge; the fourth exhibited high virus loads and significant CD4 + cell decline. Two of the DNA plus boost and three control macaques had high virus loads and associated CD4 + cell decline. Both vaccine protocols elicited antibodies and comparable helper T-cell proliferative responses to gp160. Cytokine mRNA levels in activated peripheral blood mononuclear cells (PBMC) taken at time of challenge suggested a dominant T helper (Th) 1 state in three DNA-immunized and one protein-boosted macaque, which correlated with low virus loads and high CD4 + cell counts post-challenge.

Effect of complement consumption by cobra venom factor on the course of primary infection with simian immunodeficiency virus in rhesus monkeys

Abstract: Cobra venom factor (CVF)-induced consumption of complement proteins was used to investigate the role of complement in vivo in the immunopathogenesis of simian immunodeficiency virus of macaques (SIVmac) infection in rhesus monkeys. Repeated administration of CVF was shown to deplete complement to < 5% of baseline hemolytic activity of serum complement for 10 days in a normal monkey. Three groups of SIVmac-infected animals were then evaluated: monkeys treated with CVF resulting in complement depletion from days - 1 to 10 postinfection, monkeys treated with CVF resulting in complement depletion from days 10 to 21 postinfection, and control monkeys that received no CVF. CD8+ SIVmac-specific cytotoxic T lymphocyte (CTL) generation and CD4+ T lymphocyte depletion during primary infection were not affected by CVF treatment. Viral load, assessed by measurements of plasma p27gag antigen and viral RNA, was transiently higher during the first 4 weeks following infection in the CVF-treated monkeys and the subsequent...