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Biomedical subjects

D Montefiori

Publications and source records attributed to D Montefiori.

At least 19 recordsLinked to original sources
PubMed2005

Human immunodeficiency virus type 1 subtype B ancestral envelope protein is functional and elicits neutralizing antibodies in rabbits similar to those elicited by a circulating subtype B envelope.

Human immunodeficiency virus type 1 (HIV-1) is a difficult target for vaccine development, in part because of its ever-expanding genetic diversity and attendant capacity to escape immunologic recognition. Vaccine efficacy might be improved by maximizing immunogen antigenic similarity to viruses likely to be encountered by vaccinees. To this end, we designed a prototype HIV-1 envelope vaccine using a deduced ancestral state for the env gene. The ancestral state reconstruction method was shown to be >95% accurate by computer simulation and 99.8% accurate when estimating the known inoculum used in an experimental infection study in rhesus macaques. Furthermore, the deduced ancestor gene differed from the set of sequences used to derive the ancestor by an average of 12.3%, while these latter sequences were an average of 17.3% different from each other. A full-length ancestral subtype B HIV-1 env gene was constructed and shown to produce a glycoprotein of 160 kDa that bound and fused with cells expressing the HIV-1 coreceptor CCR5. This Env was also functional in a virus pseudotype assay. When either gp160- or gp140-expressing plasmids and recombinant gp120 were used to immunize rabbits in a DNA prime-protein boost regimen, the artificial gene induced immunoglobulin G antibodies capable of weakly neutralizing heterologous primary HIV-1 strains. The results were similar for rabbits immunized in parallel with a natural isolate, HIV-1 SF162. Further design efforts to better present conserved neutralization determinants are warranted.

AIDS Vaccines↗
PubMed2001

Structured antiretroviral treatment interruptions in chronically HIV-1-infected subjects.

The risks and benefits of structured treatment interruption (STI) in HIV-1-infected subjects are not fully understood. A pilot study was performed to compare STI with continuous highly active antiretroviral therapy (HAART) in chronic HIV-1-infected subjects with HIV-1 plasma RNA levels (VL) <400 copies per ml and CD4(+) T cells >400 per microl. CD4(+) T cells, VL, HIV-1-specific neutralizing antibodies, and IFN-gamma-producing HIV-1-specific CD8(+) and CD4(+) T cells were measured in all subjects. STIs of 1-month duration separated by 1 month of HAART, before a final 3-month STI, resulted in augmented CD8(+) T cell responses in all eight STI subjects (P = 0.003), maintained while on HAART up to 22 weeks after STI, and augmented neutralization titers to autologous HIV-1 isolate in one of eight subjects. However, significant decline of CD4(+) T cell count from pre-STI level, and VL rebound to pre-HAART baseline, occurred during STI (P = 0.001 and 0.34, respectively). CD4(+) T cell counts were regained on return to HAART. Control subjects (n = 4) maintained VL <400 copies per ml and stable CD4(+) T cell counts, and showed no enhancement of antiviral CD8(+) T cell responses. Despite increases in antiviral immunity, no control of VL was observed. Future studies of STI should proceed with caution.

Adult↗
PubMed2001

An effective AIDS vaccine based on live attenuated vesicular stomatitis virus recombinants.

We developed an AIDS vaccine based on attenuated VSV vectors expressing env and gag genes and tested it in rhesus monkeys. Boosting was accomplished using vectors with glycoproteins from different VSV serotypes. Animals were challenged with a pathogenic AIDS virus (SHIV89.6P). Control monkeys showed a severe loss of CD4+ T cells and high viral loads, and 7/8 progressed to AIDS with an average time of 148 days. All seven vaccinees were initially infected with SHIV89.6P but have remained healthy for up to 14 months after challenge with low or undetectable viral loads. Protection from AIDS was highly significant (p = 0.001). VSV vectors are promising candidates for human AIDS vaccine trials because they propagate to high titers and can be delivered without injection.

AIDS Vaccines↗
PubMed2001

The stoichiometry of trimeric SIV glycoprotein interaction with CD4 differs from that of anti-envelope antibody Fab fragments.

Human and simian immunodeficiency viruses infect host lymphoid cells by binding CD4 molecules via their gp160 envelope glycoproteins. Biochemical studies on recombinant SIVmac32H (pJ5) envelope ectodomain gp140 precursor protein show that the envelope is a trimer. Using size exclusion chromatography, quantitative amino acid analysis, analytical ultracentrifugation, and CD4-based competition assay, we demonstrate that the stoichiometry of CD4 receptor-oligomeric envelope interaction is 1:1. By contrast, Fab fragments of both neutralizing and non-neutralizing monoclonal antibodies bind at a 3:1 ratio. Thus, despite displaying equivalent CD4 binding sites on each of the three gp140 protomers within an uncleaved trimer, only one site binds the soluble 4-domain human CD4 extracellular segment. The anti-cooperativity and the faster k(off) of gp140 trimer:CD4 versus gp120 monomer:CD4 interaction suggest that CD4-induced conformational change is impeded in the intact envelope. The implications of these findings for immunity against human immunodeficiency virus and simian immunodeficiency virus are discussed.

Amino Acids↗
PubMed2001

QS-21 promotes an adjuvant effect allowing for reduced antigen dose during HIV-1 envelope subunit immunization in humans.

Three separate studies were undertaken in HIV-1 uninfected persons to determine if the adjuvant QS-21 improves the magnitude or kinetics of immune responses induced by recombinant soluble gp120 HIV-1(MN) protein (rsgp120) immunization. The QS-21 was administered at two doses (50 and 100 microg), either alone or in combination with aluminum hydroxide (600 microg). At the highest doses of rsgp120 (100, 300, and 600 microg), QS-21 exerted no significant effect on either binding or neutralizing antibody titers. Antibody binding and neutralizing responses fell dramatically when rsgp120, formulated with alum alone, was given at low doses (3 and 30 microg). In contrast, antibody responses similar in titer to those in the high dose antigen groups were induced with the low dose rsgp120 formulated with QS-21. In addition, the lymphocyte proliferation and delayed type hypersensitivity skin testing were superior in the QS-21 recipients compared with the alum recipients at the low antigen doses. Moderate to severe pain was observed in majority of the volunteers receiving QS-21 formulations, and vasovagal episodes and hypertension were not infrequent. Thus, the use of QS-21 may provide a means to reduce the dose of a soluble protein immunogen.

AIDS Vaccines↗
PubMed2000

A phase II study of two HIV type 1 envelope vaccines, comparing their immunogenicity in populations at risk for acquiring HIV type 1 infection. AIDS Vaccine Evaluation Group.

Several immunogens induce HIV-specific neutralization and in vitro lymphoproliferation in adults at low HIV-1 risk, but responses in persons at high HIV-1 risk are not known. We performed a multicenter, double-blinded, adjuvant-controlled trial with two gp120 vaccines in 296 HIV-1-uninfected volunteers, including 176 reporting higher HIV-1 risk activities. The immunogens were remarkably well tolerated. After three immunizations, 210 of 241 vaccinees (87%) developed neutralizing antibodies, which persisted in 59% after 2 years. The injection drug users receiving SF-2/gp120 had decreased antibody responses relative to the lower risk groups. Envelope-specific lymphoproliferation peaked after two immunizations, and 54% of vaccinees mounted a DTH reaction to gp120 after 4 years. In summary, these immunogens have low adverse reactogenicity and induce durable antibody and T cell responses to the prototype strains. Unexpected differences in antibody responses among diverse HIV-1 risk strata lend support to the conduct of expanded phase II trials in populations other than low-risk volunteers.

AIDS Vaccines↗
PubMed2000

Effective induction of simian immunodeficiency virus-specific systemic and mucosal immune responses in primates by vaccination with proviral DNA producing intact but noninfectious virions.

We report a pilot evaluation of a DNA vaccine producing genetically inactivated simian immunodeficiency virus (SIV) particles in primates, with a focus on eliciting mucosal immunity. Our results demonstrate that DNA vaccines can be used to stimulate strong virus-specific mucosal immune responses in primates. The levels of immunoglobulin A (IgA) detected in rectal secretions of macaques that received the DNA vaccine intradermally and at the rectal mucosa were the most striking of all measured immune responses and were higher than usually achieved through natural infection. However, cytotoxic T lymphocyte responses were generally low and sporadically present in different animals. Upon rectal challenge with cloned SIVmac239, resistance to infection was observed, but some animals with high SIV-specific IgA levels in rectal secretions became infected. Our results suggest that high levels of IgA alone are not sufficient to prevent the establishment of chronic infection, although mucosal IgA responses may have a role in reducing the infectivity of the initial viral inoculum.

Animals↗
PubMed1999

Primary isolate neutralization by HIV type 1-infected patient sera in the era of highly active antiretroviral therapy.

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.

Anti-HIV Agents↗
PubMed1999

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

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 mucosal antibody regardless of the booster route. No animal in any group developed serum neutralizing antibody to homologous SIVmac251. On vaginal challenge none of the immunized groups was infected at a lesser frequency than the unimmunized controls. These data suggest that the use of microspheres in a combined parenteral and mucosal regimen is an effective method of eliciting IgG and IgA antibody at mucosal surfaces.

Animals↗
PubMed1999

A human anti-HIV autoantibody enhances EBV transformation and HIV infection.

A highly specific, human IgG mAb, F223, which reacts with both HIV-1-infected cells and uninfected lymphoid cells, has been derived. F223 reacts with gp120 but fails to neutralize viral infection. The antibody does enhance HIV-1 infection in a complement-dependent manner. The autoantigen recognized by F223 is expressed on a small percentage of T cells and NK cells and the majority of B cells. Immunoprecipitation demonstrates F223 reactivity with an as of yet unidentified 159-kDa protein in uninfected lymphoid cells. This reactivity with uninfected cells is inhibited by free gp120 demonstrating the cross-reactive nature of this antibody. The F223 light chain demonstrates strong homology to VLlambda2 family genes whereas the heavy chain is most homologous (84%) to the germline gene VH3-H.11. In vivo usage of VH3 family genes by F223 and an anti-HIV-1 (gp41) human mAb, 3D6, with related autoreactivity, suggests that VH3 sequences may be important components of potentially pathogenic human anti-HIV-1 envelope autoantibodies. F223 was isolated from an HIV-1 infected individual with lymphoma and in vitro F223 significantly enhances EBV transformation of normal B cells and increases immunoglobulin production without affecting B cell proliferation. Characterization of this antibody response may provide important insights and mechanistic information on HIV pathogenesis.

Antibodies, Blocking↗
PubMed1999

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.

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.

AIDS Vaccines↗
PubMed1998

Induction of immune responses to HIV-1 by canarypox virus (ALVAC) HIV-1 and gp120 SF-2 recombinant vaccines in uninfected volunteers. NIAID AIDS Vaccine Evaluation Group.

OBJECTIVE: To determine the ability of live attenuated canarypox virus expressing HIV antigens to induce CD8+ cytotoxic T-cell responses and to prime for neutralizing antibody responses to boosting with purified recombinant gp120 subunit vaccine. DESIGN: A prospective, double-blind, randomized, immunogenicity and safety study was conducted in healthy adults at low risk for acquiring HIV infection and who were seronegative for HIV. METHODS: CD8+ cytotoxic T-cells directed against Env or Gag expressing target cells were measured after live recombinant canarypox-HIV-1 vaccine priming (vaccine given at days 0, 7, 14 and 21). Neutralizing antibodies were measured after subunit boosting (vaccine given at days 28 and 84). RESULTS: CD8+ CTL were induced in 64% of volunteers by the live recombinant canarypox-HIV-1 vaccine. All volunteers who received two doses of subunit vaccine after live recombinant canarypox priming developed neutralizing antibodies directed against laboratory strains of HIV-1 and seven out of eight volunteers tested developed neutralizing antibodies to the primary isolate, BZ167, but to none of eight other primary isolates. Unprimed controls had low or absent neutralizing antibodies after two doses of subunit vaccine. CONCLUSIONS: The live canarypox vector was safe, stimulated cytotoxic T-cells and primed for a vigorous neutralizing antibody response upon boosting with subunit gp120 vaccine. This vaccine combination should be evaluated further for inducing protection against HIV infection.

AIDS Vaccines↗
PubMed1998

Functional and molecular characterization of human monoclonal antibody reactive with the immunodominant region of HIV type 1 glycoprotein 41.

The immunoreactivity, functional activity, and molecular features of a human monoclonal antibody (HMAb), F240, from an HIV-1-infected individual have been studied. Flow cytometric analysis demonstrated that F240 is reactive with cells infected with a broad range of laboratory isolates but not with uninfected cells. Reactivity of F240 is greatly enhanced by preincubation of infected cells with soluble CD4, and to a much lesser extent, with F105, an HMAb reactive with the CD4-binding site of gp120. This enhancement is temperature dependent, with maximum enhancement observed at 37 degrees C, and suggests that the F240 epitope may be more accessible after gp120 has bound to CD4 in vivo. Immunoblot analysis reveals antigen specificity of F240 for gp41 or its precursor gp160. F240 specificity is mapped to the immunodominant region of the gp41 ectodomain by Pepscan analysis. This epitope has been implicated in eliciting nonprotective antibodies that enhance infection in the presence of complement. Consistent with this, F240 failed to neutralize laboratory isolates and enhanced viral infection in a complement-dependent manner. The F240 VH demonstrates extensive somatic mutations compared with the product of its closest homologous germline gene VH3-3.11. Most amino acid substitutions occur in CDR2, characteristic of an antigen-driven response, and in FR3, a phenomenon observed in other anti-HIV-1 envelope HMAbs. Primary structure analysis of the F240 heavy chain revealed strong homology in the CDR domains to an HMAb (3D6) reactive with the same gp41 region, which suggests that these HMAbs could define a potential human antibody clonotype.

Amino Acid Sequence↗
PubMed1998

Simian immunodeficiency virus (SIV) envelope-specific Fabs with high-level homologous neutralizing activity: recovery from a long-term-nonprogressor SIV-infected macaque.

An antibody phage display library was constructed from RNA extracted from lymph node cells of a simian immunodeficiency virus (SIV)-infected long-term-nonprogressor macaque. Seven gp120-reactive Fabs were obtained by selection of the library against SIV monomeric gp120. Although each of the Fabs was unique in sequence, there were two distinct groups based on epitope recognition, neutralizing activity in vitro, and molecular analysis. Group 1 Fabs did not neutralize SIV and bound to a linear epitope in the V3 loop of the SIV envelope. In contrast, two of the group 2 Fabs neutralized homologous, neutralization-sensitive SIVsm isolates with high efficiency but failed to neutralize heterologous SIVmac isolates. Based on competition enzyme-linked immunosorbent assays with mouse monoclonal antibodies of known specificity, these Fabs reacted with a conformational epitope that includes domains V3 and V4 of the SIV envelope. These neutralizing and nonneutralizing Fabs provide valuable standardized and renewable reagents for studying the role of antibody in preventing or modifying SIV infection in vivo.

Amino Acid Sequence↗
PubMed1998

Recombinant vaccine-induced protection against the highly pathogenic simian immunodeficiency virus SIV(mac251): dependence on route of challenge exposure.

Vaccine protection from infection and/or disease induced by highly pathogenic simian immunodeficiency virus (SIV) strain SIV(mac251) in the rhesus macaque model is a challenging task. Thus far, the only approach that has been reported to protect a fraction of macaques from infection following intravenous challenge with SIV(mac251) was the use of a live attenuated SIV vaccine. In the present study, the gag, pol, and env genes of SIV(K6W) were expressed in the NYVAC vector, a genetically engineered derivative of the vaccinia virus Copenhagen strain that displays a highly attenuated phenotype in humans. In addition, the genes for the alpha and beta chains of interleukin-12 (IL-12), as well as the IL-2 gene, were expressed in separate NYVAC vectors and inoculated intramuscularly, in conjunction with or separate from the NYVAC-SIV vaccine, in 40 macaques. The overall cytotoxic T-lymphocyte (CTL) response was greater, at the expense of proliferative and humoral responses, in animals immunized with NYVAC-SIV and NYVAC-IL-12 than in animals immunized with the NYVAC-SIV vaccine alone. At the end of the immunization regimen, half of the animals were challenged with SIV(mac251) by the intravenous route and the other half were exposed to SIV(mac251) intrarectally. Significantly, five of the eleven vaccinees exposed mucosally to SIV(mac251) showed a transient peak of viremia 1 week after viral challenge and subsequently appeared to clear viral infection. In contrast, all 12 animals inoculated intravenously became infected, but 5 to 6 months after viral challenge, 4 animals were able to control viral expression and appeared to progress to disease more slowly than control animals. Protection did not appear to be associated with any of the measured immunological parameters. Further modulation of immune responses by coadministration of NYVAC-cytokine recombinants did not appear to influence the outcome of viral challenge. The fact that the NYVAC-SIV recombinant vaccine appears to be effective per se in the animal model that best mirrors human AIDS supports the idea that the development of a highly attenuated poxvirus-based vaccine candidate can be a valuable approach to significantly decrease the spread of human immunodeficiency virus (HIV) infection by the mucosal route.

Animals↗
PubMed1998

Viral genetic evolution in macaques infected with molecularly cloned simian immunodeficiency virus correlates with the extent of persistent viremia.

Genetic evolution of the simian immunodeficiency virus (SIV) envelope glycoprotein was evaluated in a group of six macaques (Macaca nemestrina) infected with the molecularly cloned, moderately pathogenic SIVsm62d. The extent of envelope evolution was subsequently evaluated within the context of the individual pattern of viremia and disease outcome. Two macaques in this cohort developed AIDS by 1.5 years postinoculation (progressors), whereas the remaining four macaques remained asymptomatic (nonprogressors). Compared with the nonprogressor macaques, the two progressor macaques exhibited higher persistent plasma viremia, higher homologous neutralizing antibody titers, and more extensive mutation and evolution in the V1 region of envelope. Although clearly distinct in each of these parameters from the progressors, the four nonprogressors exhibited more individual variability with respect to the extent of persistent viremia and genetic evolution of the V1 region of envelope. The extent of V1 envelope varied from no apparent V1 evolution in a macaque with good viral containment to extensive evolution in one macaque with persistent viremia. This study underscores the critical role of persistent replication in the genetic evolution of SIV.

Amino Acid Sequence↗
PubMed1997

Vaccine evaluation studies of replication-defective SIVsmB7.

Non-infectious virus-like particles of SIVsmB7 that expresses env and gag gene products but are defective in pol and vpx/vpr were assessed for their ability to induce protective immunity against infection with pathogenic SIVsmE660 in rhesus macaques. Animals were immunized in three groups: group A was primed with cell-associated SIVsmB7 and boosted with cell-free SIVsmB7; group B was primed with cell-free SIVsmB7 and boosted with cell-free SIVsmB7 conjugated to iron oxide microbeads; group C was primed with cell-free SIVsmB7 mixed with Titer Max adjuvant and boosted with cell-free SIVsmB7 mixed with SAF-M adjuvant followed by secondary boosting with cell-free SIVsmB7 conjugated to microbeads. Animals were challenged intravenously with 20 animal infectious doses of SIVsmE660 grown in rhesus peripheral blood mononuclear cells 3 weeks after final boosting. All animals became infected as evidenced by quantitative virus cultivation. Sera from immunized animals contained low-titer antibodies by ELISA and low or undetectable neutralizing antibodies on the day of challenge but strong anamnestic antibody responses were observed following challenge. Interestingly, 2 of 3 animals in group A showed evidence of transient viremia and more stable CD4 counts following challenge as compared to the other immunized animals and to non-immunized controls. Thus, immunization with cell-associated SIVsmB7 did not provide sterilizing immunity against challenge with a highly pathogenic SIV strain but might have caused virus clearance later in infection.

Animals↗