Biomedical subjects

Kenneth P Zammit

Publications and source records attributed to Kenneth P Zammit.

3 recordsLinked to original sources

PubMed2006

Antigenicity and immunogenicity of HIV-1 consensus subtype B envelope glycoproteins.

"Centralized" (ancestral and consensus) HIV-1 envelope immunogens induce broadly cross-reactive T cell responses in laboratory animals; however, their potential to elicit cross-reactive neutralizing antibodies has not been fully explored. Here, we report the construction of a panel of consensus subtype B (ConB) envelopes and compare their biologic, antigenic, and immunogenic properties to those of two wild-type Env controls from individuals with early and acute HIV-1 infection. Glycoprotein expressed from full-length (gp160), uncleaved (gp160-UNC), truncated (gp145), and N-linked glycosylation site deleted (gp160-201N/S) versions of the ConB env gene were packaged into virions and, except for the fusion defective gp160-UNC, mediated infection via the CCR5 co-receptor. Pseudovirions containing ConB Envs were sensitive to neutralization by patient plasma and monoclonal antibodies, indicating the preservation of neutralizing epitopes found in contemporary subtype B viruses. When used as DNA vaccines in guinea pigs, ConB and wild-type env immunogens induced appreciable binding, but overall only low level neutralizing antibodies. However, all four ConB immunogens were significantly more potent than one wild-type vaccine at eliciting neutralizing antibodies against a panel of tier 1 and tier 2 viruses, and ConB gp145 and gp160 were significantly more potent than both wild-type vaccines at inducing neutralizing antibodies against tier 1 viruses. Thus, consensus subtype B env immunogens appear to be at least as good as, and in some instances better than, wild-type B env immunogens at inducing a neutralizing antibody response, and are amenable to further improvement by specific gene modifications.

AIDS Vaccines↗

PubMed2006

Ancestral and consensus envelope immunogens for HIV-1 subtype C.

Immunogens based on "centralized" (ancestral or consensus) HIV-1 sequences minimize the genetic distance between vaccine strains and contemporary viruses and should thus elicit immune responses that recognize a broader spectrum of viral variants. However, the biologic, antigenic and immunogenic properties of such inferred gene products have to be validated experimentally. Here, we report the construction and characterization of the first full-length ancestral (AncC) and consensus (ConC) env genes of HIV-1 (group M) subtype C. The codon-usage-optimized genes expressed high levels of envelope glycoproteins that were incorporated into HIV-1 virions, mediated infection via the CCR5 co-receptor and retained neutralizing epitopes as recognized by plasma from patients with chronic HIV-1 subtype C infection. Guinea pigs immunized with AncC and ConC env DNA developed high titer binding, but no appreciable homologous or heterologous neutralizing antibodies. When tested by immunoblot analysis, sera from AncC and ConC env immunized guinea pigs recognized a greater number of primary subtype C envelope glycoproteins than sera from guinea pigs immunized with a contemporary subtype C env control. Mice immunized with AncC and ConC env DNA developed gamma interferon T cell responses that recognized overlapping peptides from the cognate ConC and a heterologous subtype C Env control. Thus, both AncC and ConC env genes expressed functional envelope glycoproteins that were immunogenic in laboratory animals and elicited humoral and cellular immune responses of comparable breadth and magnitude. These results establish the utility of centralized HIV-1 subtype C Env immunogens and warrant their continued evaluation as potential components of future AIDS vaccines.

AIDS Vaccines↗

PubMed2006

T-cell line for HIV drug screening using EGFP as a quantitative marker of HIV-1 replication.

The rapid increase of viral strains that are resistant to the currently available antiretroviral drugs is a threat to the success of current human immunodeficiency virus type 1 (HIV-1) treatment and emphasizes the importance of developing novel anti-HIV-1 compounds. To improve the current abilities to screen for novel HIV-1 inhibitors, here we introduce a T-cell-based reporter cell line (JLTRG-RS) that expresses both HIV-1 coreceptors, CXCR4 and CCRS, and provides the convenience of using enhanced green fluorescent protein (EGFP) as a direct and quantitative marker. Unlike previous EGFP-based reporter cell lines, JLTRG-RS cells have an unusually high dynamic signal range, sufficient for plate reader detection using a 384-well format. In this format, JLTRG-R5 cell-based infectivity assays have a Z'-factor of 0.78, which defines the assay as extremely robust and clearly amenable to high-throughput screening. The functional similarity of the JLTRG-R5 cell line and peripheral blood mononuclear cells (PBMCs) was demonstrated through the identity of the inhibitory concentrations, 50% (IC50s) for four antiretroviral compounds or neutralizing antibodies. Because EGFP can be directly and continuously quantified in cell culture, the reporter cell line requires no manipulation during assay preparation or analysis. In addition, the EGFP marker allows for data acquisition at an optimal time point by prescreening selected positive control wells using fluorescent microscopy. These characteristics make the system extremely flexible, rapid, and inexpensive. Due to its intrinsic flexibility, the JLTRG-R5 cell-based reporter system provides a powerful tool to greatly facilitate future screening for HIV-1 inhibitors.

Amides↗