Innocent Mboudjeka

Enhanced Immunogenicity of gp120 Protein When Combined with Recombinant DNA Priming To Generate Antibodies That Neutralize the JR-FL Primary Isolate of Human Immunodeficiency Virus Type 1

ABSTRACT Strategies are needed for human immunodeficiency virus type 1 vaccine development that improves the neutralizing antibody response against primary isolates of the virus. Here we examined recombinant DNA priming followed by subunit protein boosting as a strategy to generate neutralizing antibodies. Both plasmid-based and recombinant protein envelope (Env) glycoprotein immunogens were derived from a primary viral isolate, JR-FL. Serum from rabbits immunized with either gp120 or gp140 DNA vaccines delivered by gene gun inoculation followed by recombinant gp120 protein boosting was capable of neutralizing JR-FL. Neither the DNA vaccines alone nor the gp120 protein alone generated a detectable neutralizing antibody response against this virus. Neutralizing antibody responses using gp120 DNA and gp140 DNA for priming were similar. The results suggest that Env DNA priming followed by gp120 protein boosting provides an advantage over either approach alone for generating a detectable neutralizing antibody response against primary isolates that are not easily neutralized.

Polyvalent DNA prime and envelope protein boost HIV-1 vaccine elicits humoral and cellular responses and controls plasma viremia in rhesus macaques following rectal challenge with an R5 SHIV isolate

Abstract:u2002 Immunization of macaques with multivalent DNA encoding gp120 genes from HIV‐1 subtypes A, B, C and E and a gag gene followed by boosting with homologous gp120 proteins elicited strong anti‐gp120 antibodies capable of neutralizing homologous and to a lesser degree heterologous HIV‐1 isolates. Both Env‐ and Gag‐specific cell mediated immune (CMI) responses were detected in the immunized animals. Following rectal challenge with an SHIV isolate encoding HIV‐1Ba‐Lenv, plasma viremia in the infected immunized animals was significantly lower than that observed in the naïve animals. Further, one of six immunized animals was completely protected whereas all six naïve animals were infected. These results demonstrate that a vaccine based on priming with a polyvalent DNA vaccine from multiple HIV‐1 subtypes followed by boosting with homologous Env proteins elicits anti‐HIV‐1 immune responses capable of controlling rectal transmission of SHIVBa‐L.

An Engineered Saccharomyces cerevisiae Strain Binds the Broadly Neutralizing Human Immunodeficiency Virus Type 1 Antibody 2G12 and Elicits Mannose-Specific gp120-Binding Antibodies

ABSTRACT The glycan shield of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein serves as a barrier to antibody-mediated neutralization and plays a critical role in transmission and infection. One of the few broadly neutralizing HIV-1 antibodies, 2G12, binds to a carbohydrate epitope consisting of an array of high-mannose glycans exposed on the surface of the gp120 subunit of the Env protein. To produce proteins with exclusively high-mannose carbohydrates, we generated a mutant strain of Saccharomyces cerevisiae by deleting three genes in the N-glycosylation pathway, Och1, Mnn1, and Mnn4. Glycan profiling revealed that N-glycans produced by this mutant were almost exclusively Man8GlcNAc2, and four endogenous glycoproteins that were efficiently recognized by the 2G12 antibody were identified. These yeast proteins, like HIV-1 gp120, contain a large number and high density of N-linked glycans, with glycosidase digestion abrogating 2G12 cross-reactivity. Immunization of rabbits with whole Δoch1 Δmnn1 Δmnn4 yeast cells produced sera that recognized a broad range of HIV-1 and simian immunodeficiency virus (SIV) Env glycoproteins, despite no HIV/SIV-related proteins being used in the immunization procedure. Analyses of one of these sera on a glycan array showed strong binding to glycans with terminal Manα1,2Man residues, and binding to gp120 was abrogated by glycosidase removal of high-mannose glycans and terminal Manα1,2Man residues, similar to 2G12. Since S. cerevisiae is genetically pliable and can be grown easily and inexpensively, it will be possible to produce new immunogens that recapitulate the 2G12 epitope and may make the glycan shield of HIV Env a practical target for vaccine development.

A Yeast Glycoprotein Shows High-Affinity Binding to the Broadly Neutralizing Human Immunodeficiency Virus Antibody 2G12 and Inhibits gp120 Interactions with 2G12 and DC-SIGN

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein contains numerous N-linked carbohydrates that shield conserved peptide epitopes and promote trans infection by dendritic cells via binding to cell surface lectins. The potent and broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose-type oligosaccharides on the gp120 subunit of Env, revealing a conserved and highly exposed epitope on the glycan shield. To find an effective antigen for eliciting 2G12-like antibodies, we searched for endogenous yeast proteins that could bind to 2G12 in a panel of Saccharomyces cerevisiae glycosylation knockouts and discovered one protein that bound weakly in a Δpmr1 strain deficient in hyperglycosylation. 2G12 binding to this protein, identified as Pst1, was enhanced by adding the Δmnn1 deletion to the Δpmr1 background, ensuring the exposure of terminal α1,2-linked mannose residues on the D1 and D3 arms of high-mannose glycans. However, optimum 2G12 antigenicity was found when Pst1, a heavily N-glycosylated protein, was expressed with homogenous Man8GlcNAc2 structures in Δoch1 Δmnn1 Δmnn4 yeast. Surface plasmon resonance analysis of this form of Pst1 showed high affinity for 2G12, which translated into Pst1 efficiently inhibiting gp120 interactions with 2G12 and DC-SIGN and blocking 2G12-mediated neutralization of HIV-1 pseudoviruses. The high affinity of the yeast glycoprotein Pst1 for 2G12 highlights its potential as a novel antigen to induce 2G12-like antibodies.

Relative immunogenicity and protection potential of candidate Yersinia Pestis antigens against lethal mucosal plague challenge in Balb/C mice

n Summaryn n n Yersinia Pestis outer proteins, plasminogen activator protease and Yop secretion protein F are necessary for the full virulence of Yesinia pestis and have been proposed as potential protective antigens for vaccines against plague. In the current study, we used DNA immunization as a tool to study the relative protective immunity of these proteins with a standardized intranasal challenge system in mice. While the natural full-length gene sequences for most of these Y. pestis proteins did not display a good level of protein expression in vitro when delivered by a DNA vaccine vector, the overall immunogenicity of these wild type gene DNA vaccines was low in eliciting antigen-specific antibody responses and gene sequence modifications improved both of these parameters. However, even modified YopD, YopO and YscF antigens were only able to partially protect immunized mice at various levels against lethal challenge with Y. pestis KIM 1001 strain while no protection was observed with either the YopB or Pla antigens. These results demonstrate that DNA immunization is effective in screening, optimizing and comparing optimal antigen designs and immunogenicity of candidate antigens for the development of a subunit-based plague vaccine.n n

Antigen engineering can play a critical role in the protective immunity elicited by Yersinia pestis DNA vaccines.

The use of a DNA immunization approach to deliver protective antigens against Yersinia pestis (Y. pestis) has been successful in previously reported studies. In the current study, the gene designs for V and F1, two well-studied virulent factors serving as main targets for vaccine development, were altered to explore additional options in hopes of improving the protective immunity of DNA vaccines expressing these two antigens. Compared to the wild type V gene DNA vaccines, the use of codon optimized V gene sequences was effective in improving the antigen expression, titers of anti-V antibody responses, and survival against a mucosal lethal challenge. For the F1 DNA vaccine, removal of the N-terminal hydrophobic region was able to improve protective immunity. However, adding a mammalian signal peptide sequence to F1 actually led to reduced protection despite it inducing slightly higher anti-F1 antibody responses. The F1 gene can be fused with a gene coding for YscF, a newly confirmed partial protective antigen for Y. pestis, to produce DNA vaccines that express fused F1 and YscF antigens. One design, in particular, that had YscF fused to the downstream sequence of F1, produced better protection than separate F1 or YscF DNA vaccines, suggesting a potential synergistic effect between these two antigens. Findings from the above studies indicated that there are multiple approaches to optimize the protective immunity for plague DNA vaccines. Most importantly, proper antigen engineering to produce optimal antigen gene inserts in DNA vaccines can clearly play a major role in the future designs of a wide range of DNA vaccines.

Levels of N-linked glycosylation on the V1 loop of HIV-1 Env proteins and their relationship to the antigenicity of Env from primary viral isolates.

A good understanding about the structure and function of the envelope glycoprotein (Env) from primary human immunodeficiency virus-1 (HIV-1) isolates is important in facilitating the development of effective neutralizing antibody responses as a component of an effective HIV-1 vaccine. In the current study, the antigenicity of a panel of diverse HIV-1 primary Env from different clades of HIV-1 Group M was analyzed using rabbit sera produced by either 3- or 9-valent gp120 DNA vaccine formulations. Both the 3- and 9-valent gp120 DNA vaccine formulations elicited HIV-1 gp120-specific antibodies in immunized rabbits. However, we observed two levels of primary envelope antigenicity to the same set of rabbit immune sera and that the level of glycosylation, particularly in the V1 loop, may contribute to such diversity. Bioinformatics analysis on the distribution and average number of the N-linked glycosylation sites in all variable regions (V1-V5) was conducted. A linear plot demonstrated that the average number of potential N-glycosylation sites in the V1 and V4 loops correlates to the size of the loop. These data provide further evidence on the complexity of primary HIV-1 Env antigens and offers new insight into the mechanisms that HIV-1 uses to escape protective immune responses.