Proteins from blood and genital fluid isolates

The recombinant envelope protein (gp120) of the human immunodeficiency virus type 1 (HIV-1) CRF01_AE env gene isolated from the corresponding blood (rgp120F36PC) and genital fluid (rgp120-F36VC) specimens obtained from HIV infected individuals was successfully produced in both prokaryote and eukaryote cells. The yields of HIV-1 recombinant envelope proteins rgp120-F36PC and rgp120-F36VC produced in E. coli and in mammalian cells were 1.0 and 1.2, and 0.3 and 0.5 mg/ml, respectively. Antibody responses in mice immunized with rgp120-F36VC protein were not significantly higher than those with rgp120-F36PC protein. The level of antibody response in mice immunized with V3 deleted recombinant gp120 proteins from rgp120-F36VC and rgp120-F36PC was not significantly different from wild type rgp120 proteins. βstrands at the tip of the V3 loop of the HIV-1 envelope protein were predicted for the wild type genital fluid isolate but not for the wild type blood isolate. The replication capacity of both F36PC and F36VC was quite efficient. The infectivity assay of the epithelial cell line for pNL4-3/gp120F36VC was better than for pNL4-3/gp120F36PC. The extra β-strands in the V3 loop may be involved in cell tropism. drome, there is yet no effective vaccine against the virus. One of the biggest obstacles in developing an effective AIDS vaccine is the extreme difficulty in eliciting neutralizing antibodies (Nabs) that are broadly reactive against diverse HIV-1 strains (Burton et al, 1997; Parren et al 1997). The difficulty in eliciting Nabs against the virus comes from three major factors (Nyambi et al, 1998, 2000) the highly variable antigenic structure, the extensively glycosylated viral surface envelope glycoproteins, the conserved regions of the protein that binds cellular CD4 receptors and the coreceptors are obscured by variable loops (Thali et al, 1993). The poor immunogenic property of gp120 in eliciting Nabs against the virus is readily INTRODUCTION HIV-1 causes a chronic infection in humans which results in defective cellular immunity secondary to CD4 lymphocyte destruction causing AIDS (Barre-Sinoussi et al, 1983; Gallo et al, 1984; Levy et al, 1984, 1998; Ostrowski, 2005). It has been two decades since the human immunodeficiency virus type 1 (HIV-1) was identified as the etiologic agent of acquired immunodeficiency synSTRUCTURE AND FUNCTION OF HIV-1 CRF01_AE ENVELOPE PROTEINS Vol 40 No. 3 May 2009 481 apparent from the analyses of the crystal structure of the gp120 core. A three dimensional model of the crystal structure shows a significant fraction of the exposed surface of the trimeric envelope glycoprotein on the virion is occupied by carbohydrate residues and a hypervariable region (Kwong et al, 2000). To elicit Nabs that have broad cross reactivity against a large number of genetically diverse HIV-1 isolates, a vaccine candidate has to be designed so the conserved regions of the gp120 are exposed as much as possible. A number of strategies have been proposed or are being explored to elicit broadly reactive Nabs, including the use of fusion intermediates, CD4 independent envelope glycoproteins, glycosylation site mutation, and variable loop-deleted envelope proteins (Wyatt et al, 1993; Barnett et al, 2001; Kim et al, 2003). The rationale behind these approaches is to expose conserved epitopes of the protein that are common to many HIV-1 isolates by either eliminating the shield (carbohydrates or variable loops) or by capturing various transitional states of the envelope. Recently, different anatomical compartments have been the focus of attention as related to the evolution of HIV-1 in different environments. Several studies have identified the composition of the virus recovered from plasma and peripheral blood mononuclear cells (PBMC) (Simmonds et al, 1991; Livingstone et al, 1996) during HIV-1 infection. The virus was also found to have evolved into a variety of different viral sequences in the PBMC and mucosa (rectal and cervical)(Poss et al, 1995, 1998; Zhang et al, 2002). Genital secretions are the most common source of human immunodeficiency virus resulting in more than 90% of new infections transmitted heterosexually. Virus in the female genital tract may be important for both vertical (mother-tochild) and heterosexual (female-to-male) transmission. Previous studies examining portions of the HIV-1 genome have documented viral variants in the genital tract that differ from those in the blood (Delwart et al, 1998). Investigation of the genotypic and phenotypic features of HIV-1 isolates from the female genital tract is critical for the development of new therapeutic and preventive strategies to prevent heterosexual transmission. Construction of recombinant protein from HIV-1 isolates was carried out. The protein produced was immunized into mice and the antibody response was studied. We then studied isolates with the V3 loop deleted in order to evaluate the effect of a change in structure on the immune response compared with the wild type. The three dimensional structure of each isolate was predicted and compared between the wild type and the type with the V3 deletion. MATERIALS AND METHODS