We have identified several patient sera showing potent and broad HIV-1 neutralization. Using antibody adsorption and elution from selected gp120 variants, the neutralizing specificities of the two most broadly reactive sera were mapped to the primary receptor CD4-binding region of HIV-1 gp120. Novel antibodies to the CD4-binding site are elicited in some HIV-1-infected individuals, and new approaches to present this conserved region of gp120 to the immune system may result in improved vaccine immunogens.

The filovirus Ebola causes hemorrhagic fever with 70 -80% human mortality. High case-fatality rates, as well as known aerosol infectivity, make Ebola virus a potential global health threat and possible biological warfare agent. Development of an effective vaccine for use in natural outbreaks, response to biological attack, and protection of laboratory workers is a higher national priority than ever before. Coexpression of the Ebola virus glycoprotein (GP) and matrix protein (

The magnitude and durability of immune responses induced by replication-defective adenovirus serotype 5 (ADV5) vector-based vaccines were evaluated in the simian-human immunodeficiency virus/rhesus monkey model. A single inoculation of recombinant ADV5 vector constructs induced cellular and humoral immunity, but the rapid generation of neutralizing anti-Ad5 antibodies limited the immunity induced by repeated vector administration. The magnitude and durability of the immune responses elicited by these vaccines were greater when they were delivered as boosting immunogens in plasmid DNA-primed monkeys than when they were used as single-modality immunogens. Therefore, administration of ADV5-based vectors in DNA-primed subjects may be a preferred use of this vaccine modality for generating long-term immune protection.Accumulating data suggest that an effective human immunodeficiency virus (HIV) vaccine should elicit potent and durable virus-specific cellular and humoral immune responses. Vaccine-elicited immune responses can contribute to ameliorating simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus-induced disease in macaques (1,2,8,9,13,15). However, the durability of the immune responses generated by vaccination may not be adequate to provide clinical protection over a prolonged period of time. In fact, we have recently shown that the immune responses in plasmid DNA-primed rhesus monkeys that were boosted with recombinant poxviruses decayed so rapidly following the boosting immunization that no incremental clinical protection was afforded by the delivery of the live recombinant vector (14). We sought to evaluate the durability of immune responses in rhesus monkeys generated with a plasmid DNA prime/recombinant adenovirus boost vaccine.Serotype 5 human adenovirus made replication incompetent by mutation of the viral E1 and E3 genes (ADV5) has proven a safe and highly immunogenic vector in studies with laboratory animals and early-phase human clinical trials (4, 5, 17). These results have provided a rationale for advancing recombinant ADV5 into efficacy testing in human volunteers (11). However, only limited work has been done to determine the durability of the immunity that can be generated through immunization with these vaccine constructs. The present studies were performed in Indian-origin rhesus monkeys to evaluate the magnitude and persistence of the immune responses generated following recombinant ADV5 immunization. ADV5 vaccine constructs were first evaluated for immunogenicity with or without prior immunization using plasmid DNA. DNA plasmids expressing codon-optimized HIV type 1 (HIV-1) and SIV immunogens were made synthetically, using a method that has been previously described (10). The full-length synthetic SIVmac239 gag-pol-nef gene encoding a fusion protein was cloned in the mammalian expression vector pVR1012 under the control of the cytomegalovirus immediateearly enhancer, promoter, and first intron. The pVR1012-HIV-1 89.6P Env plasmid expresses a modified form of the env ...

This study examined the biologic responses of transgenic mice expressing human leukocyte antigen (HLA)-DR3 and human CD4 molecules, in the absence of murine major histocompatibility complex (MHC) class II molecules (Ab(0)), to staphylococcal enterotoxins (SEs) and evaluated protective immunity of a nonsuperantigen form of SEB against wild-type holotoxin. HLA-DR3 transgenic mice responded to several log lower concentrations of SEs and secreted higher levels of proinflammatory cytokines than did wild-type mice. Vaccination of transgenic mice with a nonsuperantigenic form of SEB induced high levels of neutralizing anti-SEB antibodies, which protected the mice from a surge in proinflammatory cytokine secretion after SEB challenge. The humanlike responses of the transgenic mice to SEs support the hypothesis that these mice represent an appropriate model to examine vaccines and therapeutics against SEs. This is thought to be the first report of examination of a vaccine against SEB in the context of human MHC class II receptors.

Immunization with recombinant serotype 5 adenoviral (rAd5) vectors or a combination of DNA plasmid priming and rAd5 boosting is known to elicit potent immune responses. However, little data exist regarding these immunization strategies and the development of anti-human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies. We used DNA plasmids and rAd5 vectors encoding the HIV-1 89.6P or chimeric HxB2/BaL envelope glycoprotein to immunize macaque monkeys. A single rAd5 immunization elicited anti-Env antibody responses, but there was little boosting with subsequent rAd5 immunizations. In contrast, rAd5 boosting of DNA-primed monkeys resulted in a rapid rise in antibody titers, including the development of anti-HIV-1 neutralizing antibodies. The potency and breadth of neutralization were evaluated by testing plasma against a panel of 14 clade B primary isolates. Moderate levels of plasma neutralizing activity were detected against about one-third of the viruses tested, and immunoglobulin G fractionation demonstrated that virus neutralization was antibody mediated. After a challenge with a chimeric simian-human immunodeficiency virus (SHIV89.6P), an anamnestic neutralizing antibody response was observed, although the breadth of the response was limited to the subset of viruses that were neutralized after the primary immunization. These data are the first detailed description of the anti-HIV-1 neutralizing antibody response in nonhuman primates elicited by DNA and rAd5 immunization. In addition to the well-established ability of DNA priming and rAd5 boosting to elicit potent anti-HIV-1 cellular immune responses, this immunization strategy elicits anti-HIV-1 neutralizing antibodies and therefore can be used to study novel Env immunogens designed to elicit more potent neutralizing antibodies.

Staphylococcal enterotoxins (SEs) belong to a large group of bacterial exotoxins that cause severe immunopathologies, especially when delivered as an aerosol. SEs elicit the release of lethal amounts of cytokines by binding to major histocompatibility complex (MHC) class II and cross-linking susceptible T-cell receptors. Efforts to develop effective therapeutic strategies to protect against SEs delivered as an aerosol have been hampered by the lack of small animal models that consistently emulate human responses to these toxins. Here, we report that human leukocyte antigen-DQ8 (HLA-DQ8) transgenic (Tg) mice, but not littermate controls, succumbed to lethal shock induced by SEB aerosols without potentiation. Substantial amounts of perivascular edema and inflammatory infiltrates were noted in the lungs of Tg mice, similar to the pathology observed in nonhuman primates exposed by aerosol to SEB. Furthermore, the observed pathologies and lethal shock correlated with an upsurge in proinflammatory cytokine mRNA gene expression in the lungs and spleens, as well as with marked increases in the levels of proinflammatory circulating cytokines in the Tg mice. Unlike the case for littermate controls, telemetric evaluation showed significant hypothermia in Tg mice exposed to lethal doses of SEB. Taken together, these results show that this murine model will allow for the examination of therapeutics and vaccines developed specifically against SEB aerosol exposure and possibly other bacterial superantigens in the context of human MHC class II receptors.Staphylococcus aureus and group A streptococci are responsible for a wide range of mild to life-threatening infections, including scarlet fever, pharyngitis, dermatitis, infectious arthritis, and toxic shock syndrome (2, 13, 18, 25, 31). These pathogenic bacteria use several virulence mechanisms to enhance their toxicity after infection, including the M protein, diffusible enzymes (e.g., DNase), and streptolysin. Furthermore, many strains of S. aureus produce bacterial superantigens (BSAgs), which exert a series of critical, negative immunological effects on the host. Specifically, BSAgs bind to major histocompatibility complex (MHC) class II molecules and form a ternary complex with receptive variable ␤ chains of T-cell antigen receptors. After binding, BSAg-stimulated T cells are eliminated by a Fas/Fas-ligand-mediated apoptosis or, alternatively, enter a state of specific nonresponsiveness (anergy), which may last for several months. Furthermore, BSAgs may exacerbate subclinical viral infections by removing activated T cells from their normal role in clearing invasive organisms.Mice are naturally insensitive to BSAg-induced lethal shock (23,24,32). In order to overcome the natural insensitivity of mice to staphylococcal enterotoxins (SEs), sublethal amounts of lipopolysaccharide (LPS) have been used to potentiate the lethal effects of BSAgs (7,32). Although the exact mechanism of LPS induction is not known, it has been shown that lethality is dependent on the expression of mo...

Although the B clade of human immunodeficiency virus type 1 (HIV-1) envelopes (Env) includes five highly variable regions, each of these domains contains a subset of sequences that remain conserved. The V3 loop has been much studied for its ability to elicit neutralizing antibodies, which are often restricted to a limited number of closely related strains, likely because a large number of antigenic structures are generated from the diverse amino acid sequences in this region. Despite these strain-specific determinants, subregions of V3 are highly conserved, and the effects of different portions of the V3 loop on Env tropism and immunogenicity have not been well delineated. For this report, selective deletions in V3 were introduced by shortening of the stem of the V3 loop. These mutations were explored in combination with deletions of selected V regions. Progressive shortening of the stem of V3 abolished the immunogenicity as well as the functional activity of HIV Env; however, two small deletions on both arms of the V3 stem altered the tropism of the dualtropic 89.6P viral strain so that it infected only CXCR4 ؉ cells. When this smaller deletion was combined with removal of the V1 and V2 loops and used as an immunogen in guinea pigs, the antisera were able to neutralize multiple independent clade B isolates with a higher potency. These findings suggest that highly conserved subregions within V3 may be relevant targets for eliciting neutralizing antibody responses, affecting HIV tropism, and increasing the immunogenicity of AIDS vaccines.

No abstract