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Cheng Cheng, Lingshu Wang,
Jason G D Gall,
Martha Nason,
Richard M Schwartz,
M Juliana McElrath,
Steven C Derosa,
John Hural,
Lawrence Corey,
Susan P Buchbinder,
Gary J Nabel
PLoS ONE 01/2012; 7(5). · 4.09 Impact Factor
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Cheng Cheng, Lingshu Wang,
Jason G D Gall,
Martha Nason,
Richard M Schwartz,
M Juliana McElrath,
Steven C DeRosa,
John Hural,
Lawrence Corey,
Susan P Buchbinder,
Gary J Nabel
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ABSTRACT: The Step trial raised the possibility that uncircumcised men with pre-existing Ad5 neutralizing antibodies carried an increased risk of HIV infection after vaccination. Thus, understanding Ad seropositivity in humans is important to the development of an AIDS vaccine. Here, we analyze the impact of different Ad5-specific neutralizing antibodies on immune function and clinical outcome.
Ad seropositivity in the Step trial volunteers was analyzed using chimeric rAd5/35 vectors to characterize their specificity for Ad5 fiber and non-fiber external (capsid) proteins. Immune responses and HIV seropositivity were correlated with the specificity of Ad5-neutralizing antibodies. Neutralizing antibodies induced by the vaccine in Ad5 seronegative subjects were directed preferentially to Ad5 capsid proteins, although some fiber-neutralizing antibodies could be detected. Pre-vaccination Ad5 serostatus did not affect the capsid-directed response after three vaccinations. In contrast, anti-fiber antibody titers were significantly higher in volunteers who were Ad5 seropositive prior to vaccination. Those Ad5 seropositive subjects who generated anti-capsid responses showed a marked reduction in vaccine-induced CD8 responses. Unexpectedly, anti-vector immunity differed qualitatively in Ad5 seropositive participants who became HIV-1 infected compared to uninfected case controls; Ad5 seropositive participants who later acquired HIV had lower neutralizing antibodies to capsid. Moreover, Ad35 seropositivity was decreased in HIV-infected subjects compared with uninfected case controls, while seroprevalence for other serotypes including Ad14, Ad28 and Ad41 was similar in both groups.
Together, these findings suggest that the case subjects were less immunologically responsive prior to infection. Subjects infected during the Step trial had qualitative differences in immunity that increased their risk of HIV-1 infection independent of vaccination.
PLoS ONE 01/2012; 7(4):e33969. · 4.09 Impact Factor
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ABSTRACT: Effective vaccines for human immunodeficiency virus type 1 (HIV-1) will likely need to stimulate protective immunity in the intestinal mucosa, where HIV-1 infection causes severe CD4(+) T-cell depletion. While replication-competent recombinant adenovirus (rAd) vectors can stimulate adenovirus-specific mucosal immunity after replication, oral delivery of replication-defective rAd vectors encoding specific immunogens has proven challenging. In this study, we have systematically identified barriers to effective gut delivery of rAd vectors and identified sites and strategies to induce potent cellular and humoral immunity. Vector-mediated gene transfer by rAd5 was susceptible to low-pH buffer, gastric and pancreatic proteases, and extracellular mucins. Using ex vivo organ explants, we found that transduction with rAd5 was highest in the ileum and colon among all intestinal segments. Transgene expression was 100-fold higher after direct surgical introduction into the ileum than after oral gavage, with rAd5 showing greater potency than the rAd35 or the rAd41 vector. A single immunization of rAd5 encoding HIV-1 gp140B to the ileum stimulated potent CD8(+) T-cell responses in the intestinal and systemic compartments, and these responses were further enhanced by intramuscular rAd5 boosting. These studies suggest that induction of primary immune responses by rAd5 gut immunization and subsequent systemic boosting elicits potent antigen-specific gut mucosal responses.
Journal of Virology 06/2009; 83(14):7166-75. · 5.40 Impact Factor
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ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) infection is characterized by the rapid onset of intestinal T-cell depletion that initiates the progression to AIDS. The induction of protective immunity in the intestinal mucosa therefore represents a potentially desirable feature of a preventive AIDS vaccine. In this study, we have evaluated the ability of an enteric adenovirus, recombinant adenovirus 41 (rAd41), to elicit intestinal and systemic immune responses by different immunization routes, alone or in combination with rAd5. rAd41 expressing HIV envelope (Env) protein induced cellular immune responses comparable to those of rAd5-based vectors after either a single intramuscular injection or a DNA prime/rAd boost. Oral priming with rAd41-Env followed by intramuscular boosting with rAd5-Env stimulated a more potent CD8(+) T-cell response in the small intestine than the other immunization regimens. Furthermore, the direct injection of rAd41-Env into ileum together with intramuscular rAd5-Env boosting increased Env-specific cellular immunity markedly in mucosal as well as systemic compartments. These data demonstrate that heterologous rAd41 oral or ileal priming with rAd5 intramuscular boosting elicits enhanced intestinal mucosal cellular immunity and that oral or ileal vector delivery for primary immunization facilitates the generation of mucosal immunity.
Journal of Virology 12/2008; 83(2):748-56. · 5.40 Impact Factor
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ABSTRACT: HIV-1 envelope glycoprotein gp120 induces, independently of infection, the release of proinflammatory cytokines, including IL-1beta from macrophages, that are implicated in the pathogenesis of HIV-associated dementia. However, the signal transduction pathways involved have not been fully defined. Previously, our laboratory reported that soluble gp120 activates multiple protein kinases in primary human monocyte-derived macrophages, including the Src family kinase Lyn, PI3K, and the focal adhesion-related proline-rich tyrosine kinase Pyk2. In this study we showed that gp120 induces IL-1beta release from macrophages in a time- and concentration-dependent manner through binding to the chemokine receptor CCR5 and coupling to G(i)alpha protein. Using pharmacological inhibitors and small interfering RNA gene knockdown, we demonstrated that concomitant activation of Lyn, Pyk2, and class IA PI3K are required for gp120-induced IL-1beta production. By coimmunoprecipitation and immunofluorescence confocal microscopy, we showed that CCR5 activation by gp120 triggered the assembly of a signaling complex involving endogenous Lyn, PI3K, and Pyk2 and is associated with PI3K and Pyk2 translocation from the cytoplasm to the membrane where they colocalized with Lyn. Finally, we demonstrated that virion-associated gp120 induced similar response, as structurally intact whole virions also triggered IL-1beta release and re-localization of PI3K and Pyk2. This study identifies a novel signaling mechanism for HIV-1-induced IL-1beta production by primary human macrophages that may be involved in the neuropathogenesis of HIV-associated dementia.
The Journal of Immunology 06/2008; 180(10):6675-84. · 5.79 Impact Factor
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ABSTRACT: Macrophages are important targets for HIV-1, and R5X4 strains play a central role in pathogenesis, especially in late-stage patients who may receive the fusion inhibitor T20 (enfuvirtide). Sensitivity to T20 varies markedly among HIV-1 strains and is influenced by viral and cellular factors that affect Env/CD4/coreceptor interactions. We addressed the relation between T20 inhibition and the pathway by which R5X4 HIV-1 infects primary macrophages, which express both coreceptors. In U87/CD4/coreceptor cells, T20 sensitivity for entry through CCR5 and CXCR4 was correlated. In macrophages, the proportion of total entry mediated by each coreceptor differed among isolates. Neither pathway was uniformly more or less sensitive to T20, however, nor did the proportion of entry mediated by each coreceptor predict T20 sensitivity. T20 sensitivity for macrophage infection overall correlated modestly with that for entry through CCR5 but not through CXCR4; however, unlike U87 cells, sensitivity of entry through CCR5 and CXCR4 was not correlated. These results suggest that strain-specific factors influence R5X4 T20 sensitivity regardless of the coreceptor used, an absence of systematic differences in efficiency by which R5X4 strains use the 2 coreceptors, and that efficiency and kinetics of interactions with CCR5 are central determinants of macrophage entry even when both pathways are utilized.
JAIDS Journal of Acquired Immune Deficiency Syndromes 04/2008; 47(3):285-92. · 4.43 Impact Factor
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ABSTRACT: Recently we demonstrated that attachment of three copies of murine C3d (muC3d) to the E2 envelope protein of bovine viral diarrhea virus results in a 10,000-fold increase in the immunogenicity of E2. Here we describe the cloning of the bovine homolog of C3d (boC3d), construction of an E2-boC3d expression cassette and expression and purification of the E2-boC3d fusion protein. We then show that E2, when coupled to boC3d, exhibits greatly enhanced immunogenicity. Thus, boC3d represents the second mammalian C3d homolog, thus far, shown to enhance the immunogenicity of a protein to which it has been coupled. Although the primary sequence of boC3d differs from muC3d by about 19%, we were able to demonstrate the enhanced immunogenicity of E2-boC3d using mice. The ability of boC3d to function in mice provides a less costly and more convenient animal model than cattle for the preliminary evaluation of E2-boC3d and other bovine antigen-boC3d fusion proteins.
Developmental & Comparative Immunology 02/2005; 29(10):907-15. · 3.27 Impact Factor
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ABSTRACT: The use of DNA and protein subunit vaccines in animals provides an opportunity to introduce vaccines that are arguably the safest that can be developed. For that reason, considerable effort is under way to devise methods of enhancing the immunogenicity of such vaccines. Seven years ago it was shown that fusing complement fragment C3d to hen egg lysozyme (HEL) enhanced the immunogenicity of HEL 10,000-fold. Based on this observation, we decided to evaluate the effect of C3d on the immunogenicity of the E2 protein of bovine viral diarrhea virus (BVDV). E2 is the major target of neutralizing antibody during BVDV infection. To test the effect of C3d on E2 immunogenicity, expression cassettes encoding a secreted form of E2 alone (E2s) or E2 fused to three copies of murine C3d (E2s-C3d) were constructed. The proteins were purified from the supernatants of transfected cells and used to immunize mice. The immune response was monitored by an enzyme-linked immunosorbent assay (ELISA) for E2s-specific antibody and by a virus neutralization test. The ELISA results indicated that the E2s-C3d protein is 10,000-fold more immunogenic than the E2s protein alone. The maximum primary immune response was elicited with <0.1 microg of E2s-C3d protein without an adjuvant. In addition, we have shown for the first time that high levels of anti-E2s and neutralizing antibodies can be elicited when this same low concentration of E2s-C3d is used to both prime and boost the immune response. We conclude that the E2s-C3d fusion protein has significant potential as a subunit vaccine against BVDV infection.
Journal of Virology 03/2004; 78(4):1616-22. · 5.40 Impact Factor
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ABSTRACT: Bovine viral diarrhea virus (BVDV) is a ubiquitous pathogen of cattle with a world-wide distribution. Recently, the possibility of using recombinant virus vectors to immunize cattle against selected BVDV genes has gained widespread interest. Among the virus vectors tested, bovine herpesvirus-1 (BHV1) provides many unique advantages. However, results of recent studies have raised the possibility that the codon usage pattern required for optimal expression in a BHV1-infected cell may be incompatible with the codon usage pattern of BVDV. If true, use of BHV1 to express BVDV proteins would require construction of synthetic BVDV genes that have been modified to resemble the codon pattern of BHV1. To explore this possibility, we constructed a BHV1 recombinant containing the genomic form of the BVDV (NADL) E2 ORF and compared expression of the E2 protein with that of the endogenous BHV1 gD protein. We observed that E2 was expressed at a significant rate compared to that of the gD protein. We conclude that codon usage problems are unlikely to constitute a serious problem for expression of BVDV proteins in BHV1 vectors.
Virus Genes 09/2003; 27(1):83-91. · 1.85 Impact Factor
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ABSTRACT: Recently, the possibility of using virus vectors to immunize cattle against selected bovine viral diarrhea virus (BVDV) genes has gained widespread interest. However, when we attempted to express the E2 protein from type 2 (890 strain) BVDV in a bovine herpesvirus 1 (BHV1) vector, we observed that expression was poor. This often happens when genes from a cytoplasmic virus are expressed in the cell nucleus. To counter this effect, we attempted to enhance expression by a strategy employed by viruses. RNAs of retroviruses and hepadnaviruses contain cis-acting elements that facilitate expression of RNAs that otherwise are degraded or retained within the nucleus. In Mason-Pfizer monkey virus, the required RNA sequence element is known as a constitutive transport element (CTE). A related element from woodchuck hepatitis virus is known as the woodchuck posttranscriptional regulatory element (WPRE). We tested the ability of the CTE, the WPRE, and introns to enhance expression of E2. All three elements stimulated expression of E2 from plasmids. The combination of the WPRE and an intron yielded the highest level of E2 expression in plasmids. However, when E2 was expressed from a BHV1 vector, the presence of an intron was inhibitory. In contrast, the WPRE was very efficient at stimulating E2 expression from a BHV1 vector. This result represents the first expression of a type 2 BVDV E2 protein from a mammalian virus vector and raises the possibility that the WPRE may provide a general method of enhancing foreign gene expression from BHV1 and other herpesvirus vectors.
Journal of Virology 09/2003; 77(16):8775-82. · 5.40 Impact Factor
