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Abstract Background In order to induce a potent and cross-reactive neutralizing antibody (nAb), an effective envelope immunogen is crucial for many viral vaccines, including the vaccine for the human immunodeficiency virus (HIV). The Chinese equine infectious anemia virus (EIAV) attenuated vaccine has controlled the epidemic of this virus after its vaccination in over 70 million equine animals during the last 3 decades in China. Data from our past studies demonstrate that the Env protein of this vaccine plays a pivotal role in protecting horses from both homologous and heterogeneous EIAV challenges. Therefore, the amino acid sequence information from the Chinese EIAV attenuated vaccine, in comparison with the parental wild-type EIAV strains, was applied to modify the corresponding region of the envelope glycoprotein of HIV-1 CN54. The direction of the mutations was made towards the amino acids conserved in the two EIAV vaccine strains, distinguishing them from the two wild-type strains. The purpose of the modification was to enhance the immunogenicity of the HIV Env. Results The induced nAb by the modified HIV Env neutralized HIV-1 B and B'/C viruses at the highest titer of 1:270. Further studies showed that a single amino acid change in the C1 region accounts for the substantial enhancement in induction of anti-HIV-1 neutralizing antibodies. Conclusions This study shows that an HIV envelope modified by the information of another lentivirus vaccine induces effective broadly neutralizing antibodies. A single amino acid mutation was found to increase the immunogenicity of the HIV Env.

Antiretroviral therapy (ART) significantly reduced the CD8+ cell noncytotoxic anti-HIV response in 12 HIV-1-infected subjects (P < 0.0001). In separate experiments, CD8+ cells from long-term survivors were cocultured with HIV-infected CD4+ cells using varying concentrations of anti-HIV drugs. The antiviral function of CD8+ cells from 4 of the 14 LTSs was reduced with exposure to 10 μM of nevirapine (P < 0.05). The antiviral activity of CD8+ cells from 2 LTSs was inhibited by 5 μM of zidovudine. These studies indicate that nevirapine and probably zidovudine can inhibit the anti-HIV activity of CD8+ cells and thus could influence the effectiveness of antiretroviral therapy. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To develop an effective HIV vaccine strategy that can induce cross-reactive neutralizing antibody. Methods: Codon-optimized gp140 and gp145 env genes derived from HIV-1cn54, a CRF07 B′/C recombinant strain, were constructed as DNA and recombinant Tiantan vaccinia (rTV) vaccines. The effect of heterologous immunization with gp140 and gp145 was tested in mice and guinea pigs. T cell responses were detected using the IFN-γ ELISPOT assay. A panel of primary isolates of clade B′ and B′/C HIV-1 and TZM-bl cells was used to determine the neutralizing activity of immunized sera. Results: The neutralizing antibodies (NAbs) induced by the heterologous immunogen immunization neutralized all HIV-1 B′ and B′/C primary isolates in the guinea pig model. Gp145 and gp140 heterologous prime-boost induced the best neutralizing antibody response with a broad neutralizing spectrum and the highest titer of 1:270 at 6 weeks after the last inoculation. However, the T cell response to HIV-1 peptides was significantly weaker than the gp145+gp145 homologous prime-boost. Conclusions: This heterologous prime-boost immunization strategy could be used to design immunogen-generating broad neutralizing antibodies against genetic variance pathogens. [Copyright &y& Elsevier]

Abstract: Electroporation, a non-virus-mediated gene transfection method, has traditionally had poor outcomes with low gene transfection efficiency and poor cellular viability, particularly in primary human lymphocytes. Herein we have optimized the electroporation conditions for primary CD8+ cells resulting in a maximum rate of 81.3%, and a mean transfection efficiency of 59.6%. After removal of dead cells, the viability of transfected primary CD8+ cells was greater than 90%, similar to untransfected controls. Using this procedure, primary human CD8+ cells transfected with an interferon α8 plasmid produced fluids that inhibited HIV-1 replication by >95%. This transfection protocol is useful for transfection of other primary blood cells, such as CD4+ T cells, and for studying the function of genes in primary human blood cells instead of cell lines. The transfection procedure also has potential application in gene therapy clinical trials to treat diseases utilizing transfected primary human cells. [Copyright &y& Elsevier]

Depletion of T regulatory cells (Tregs) in the tumor microenvironment is a promising cancer immunotherapy strategy. Current approaches for depleting Tregs are limited by lack of specificity and concurrent depletion of anti-tumor effector T cells. The transcription factor forkhead box p3 (Foxp3) plays a central role in the development and function of Tregs and is an ideal target in Tregs, but Foxp3 is an intracellular, undruggable protein to date. We have generated a T cell receptor mimic antibody, "Foxp3-#32," recognizing a Foxp3-derived epitope in the context of HLA-A*02:01. The mAb Foxp3-#32 selectively recognizes CD4 + CD25 + CD127low and Foxp3 + Tregs also expressing HLA-A*02:01 and depletes these cells via antibody-mediated cellular cytotoxicity. Foxp3-#32 mAb depleted Tregs in xenografts of PBMCs from a healthy donor and ascites fluid from a cancer patient. A TCRm mAb targeting intracellular Foxp3 epitope represents an approach to deplete Tregs. [ABSTRACT FROM AUTHOR]

Abstract: Equine Infectious Anemia Virus (EIAV), like other lentiviruses, has a transmembrane glycoprotein with an unusually long cytoplasmic tail (CT). Viral envelope (Env) proteins having CT truncations just downstream the putative membrane-spanning domain (PMSD) are assumed to exist among all wild-type budded virions, and also in some cell-adapted strains. To determine whether CT-truncated Env proteins can cause particularly deleterious effects on the Env expressing cells and/or their neighboring cells, plasmids encoding codon-optimized env gene including full-length (pE863) or CT-truncated (pE686* and pE676*) were transiently transfected into 293T cells, respectively. Data from intracellular protein expression and cell death assays revealed that CT-truncated Env, compared to full-length Env, not only induced comparable apoptosis, but also caused much more intensive mitochondria-mediated necrosis that could simultaneously induce significant decrease of intracellular protein expression in the Env expressing cells. Moreover, results from flow cytometric analysis showed that mitochondrial depolarization preceded the caspase activation in cells no matter which env construct was delivered, and indicated that both full-length and CT-truncated Env proteins share a common intrinsic mitochondrial pathway to induce apoptosis. Our results partially elucidate the mechanisms underlying cell death resulting from EIAV pathogenesis. [Copyright &y& Elsevier]

Antiretroviral therapy (ART) significantly reduced the CD8 cell noncytotoxic anti-HIV response in 12 HIV-1-infected subjects (P < 0.0001). In separate experiments, CD8(+) cells from long-term survivors were cocultured with HIV-infected CD4(+) cells using varying concentrations of anti-HIV drugs. The antiviral function of CD8(+) cells from 4 of the 14 LTSs was reduced with exposure to 10 μM of nevirapine (P < 0.05). The antiviral activity of CD8(+) cells from 2 LTSs was inhibited by 5 μM of zidovudine. These studies indicate that nevirapine and probably zidovudine can inhibit the anti-HIV activity of CD8(+) cells and thus could influence the effectiveness of antiretroviral therapy.

It remains a great challenge to develop an effective HIV vaccine against the most prevalent HIV-1 clade, B'/C recombinant, in China. Our objective was to test the influence of a new modification of the V1/V2 loops of HIV-1(CN54) gp140 on the immunogenicity of Env. HIV-1(CN54) gp140 was deglycosylated by replacing all six N residues in V1/V2 loops with six Q residues (gp140dG) or partially deleted on V1/V2 loops (gp140dV). gp140, gp140dG, and gp140dV were transferred into plasmid vector and recombinant Tiantan vaccinia (rTTV) vector to generate three DNA vaccines and three rTTV vaccines for vaccination of female BALB/c mice in a prime-boost regimen. An Elispot assay was used to read out the T cell immunity and ELISA and a poly-l-leucine (PLL) ELISA was employed to assess humoral immune responses. Surprisingly, gp140dV (1570 +/- 1569 SFCs/10(6) splenocytes) and gp140dG (731 +/- 471 SFCs/10(6) splenocytes) could elicit significantly higher Env-specific T cells than gp140 (224 +/- 140 SFCs/10(6) splenocytes). Three T cell epitopes were newly identified in BALB/c mice at the N terminus of C1, C terminus of C4, and N terminus of HR, respectively. Env-specific binding antibodies and linear antibodies elicited by gp140 tended to be higher than that stimulated by gp140dG and gp140dV but did not reach statistical difference. Our data demonstrated that the deglycosylation and partial deletion of V1/V2 loops of B'/C recombinant gp140 could lead to improvement of specific T cell immune responses.

To develop an effective vaccine against the most prevalent HIV strain "B'/C recombinant" in China, we compared the immunogenicity of B'/C-derived gp140 and gp145. The codon optimized gp140 and gp145 env gene derived from CN54, an ancestor-like B'/C recombinant strain, were synthesized and cloned into a plasmid as DNA vaccines, designated as pDRVISV140 and pDRVISV145, respectively. BALB/c mice were inoculated three times at week 0, 2, and 4 and sacrificed at week 7. Both T cell immunity and humoral immunity were determined. The mock vector pDRVISV1.0 carrying no HIV immunogen was included as control. Our data showed that B'/C recombinant-derived gp145 mounted stronger T cell and broader linear antibody but less binding antibody immune responses than gp140 did. Though both gp145 and gp140 raised neutralization antibodies against laboratory-adapted strain SF33, both failed to neutralize B' or B'/C clade primary strains. Overall, this is the first time the immunogenicity of B'/C recombinant-derived gp140 and gp145 was examined and compared; our data prefer B'/C-derived gp145 to gp140 as an HIV vaccine immunogen. The failure to induce neutralization antibodies against primary isolates indicates that it is insufficient to enhance the immunogenicity of conserved epitopes by simply employing gp145 or gp140; strategies to enhance antibody responses against conserved epitopes should be explored further.