Quantitative effect of suboptimal codon usage on translational efficiency of mRNA encoding HIV-1 gag in intact T cells.

BACKGROUND: The sequences of wild-isolate strains of Human Immunodeficiency Virus-1 (HIV-1) are characterized by low GC content and suboptimal codon usage. Codon optimization of DNA vectors can enhance protein expression both by enhancing translational efficiency, and by altering RNA stability and export. Although gag codon optimization is widely used in DNA vectors and experimental vaccines, the actual effect of altered codon usage on gag translational efficiency has not been quantified. METHODOLOGY AND PRINCIPAL FINDINGS: To quantify translational efficiency of gag mRNA in live T cells, we transfected Jurkat cells with increasing doses of capped, polyadenylated synthetic mRNA corresponding to wildtype or codon-optimized gag sequences, measured Gag production by quantitative ELISA and flow cytometry, and estimated the translational efficiency of each transcript as pg of Gag antigen produced per microg of input mRNA. We found that codon optimization yielded a small increase in gag translational efficiency (approximately 1.6 fold). In contrast when cells were transfected with DNA vectors requiring nuclear transcription and processing of gag mRNA, codon optimization resulted in a very large enhancement of Gag production. CONCLUSIONS: We conclude that suboptimal codon usage by HIV-1 results in only a slight loss of gag translational efficiency per se, with the vast majority of enhancement in protein expression from DNA vectors due to altered processing and export of nuclear RNA.

A viral CTL escape mutation leading to immunoglobulin-like transcript 4-mediated functional inhibition of myelomonocytic cells.

Viral mutational escape can reduce or abrogate recognition by the T cell receptor (TCR) of virus-specific CD8+ T cells. However, very little is known about the impact of cytotoxic T lymphocyte (CTL) epitope mutations on interactions between peptide-major histocompatibility complex (MHC) class I complexes and MHC class I receptors expressed on other cell types. Here, we analyzed a variant of the immunodominant human leukocyte antigen (HLA)-B2705-restricted HIV-1 Gag KK10 epitope (KRWIILGLNK) with an L to M amino acid substitution at position 6 (L6M), which arises as a CTL escape variant after primary infection but is sufficiently immunogenic to elicit a secondary, de novo HIV-1-specific CD8+ T cell response with an alternative TCR repertoire in chronic infection. In addition to altering recognition by HIV-1-specific CD8+ T cells, the HLA-B2705-KK10 L6M complex also exhibits substantially increased binding to the immunoglobulin-like transcript (ILT) receptor 4, an inhibitory MHC class I-specific receptor expressed on myelomonocytic cells. Binding of the B2705-KK10 L6M complex to ILT4 leads to a tolerogenic phenotype of myelomonocytic cells with lower surface expression of dendritic cell (DC) maturation markers and co-stimulatory molecules. These data suggest a link between CTL-driven mutational escape, altered recognition by innate MHC class I receptors on myelomonocytic cells, and functional impairment of DCs, and thus provide important new insight into biological consequences of viral sequence diversification.

Response to hepatitis B vaccine in HIV-1-positive subjects who test positive for isolated antibody to hepatitis B core antigen: implications for hepatitis B vaccine strategies.

BACKGROUND: Whether human immunodeficiency virus type 1 (HIV-1)-positive subjects who test positive for isolated antibody to hepatitis B core antigen (anti-HBc) should be vaccinated with hepatitis B vaccine is not certain. Development of an anamnestic response after vaccination would suggest previous hepatitis B virus (HBV) infection, in which case vaccination is not necessary. METHODS: Sixty-nine HIV-1-positive subjects who tested negative for hepatitis B surface antigen (HBsAg) and antibody to HBsAg (anti-HBs) received vaccination with standard hepatitis B vaccine. Twenty-nine subjects (42%) tested positive for anti-HBc, and 40 (58%) tested negative for anti-HBc. An anamnestic response was defined as an anti-HBs titer of >or=10 IU/L within 4 weeks of the first vaccination. RESULTS: The overall anamnestic response rate was 16% and was not significantly different between subjects who tested positive for anti-HBc (24%) and those who tested negative for anti-HBc (10%) before vaccination (P=.18). Approximately 50% of subjects who tested positive for anti-HBc also tested positive for antibody to hepatitis Be antigen (anti-HBe). The anamnestic response rate was higher in subjects who tested positive for both anti-HBc and anti-HBe (43%) than in subjects who tested positive for anti-HBc but negative for anti-HBe (7%) (P=.035). After a complete series of vaccinations, HIV-1/hepatitis C virus (HCV)-coinfected subjects were less likely to achieve high anti-HBs titers than were subjects infected with HIV-1 alone. CONCLUSIONS: After hepatitis B vaccination, the anamnestic response rate in HIV-1-positive subjects who tested positive for isolated anti-HBc but negative for anti-HBe was low and was comparable to that in subjects who tested negative for anti-HBc. This finding suggests that testing for anti-HBc alone may not be a reliable assessment of protection from HBV infection. HIV-1/HCV coinfection may be associated with impaired responses to hepatitis B vaccine, and evaluation of strategies to improve immunogenicity of the vaccine in such individuals is warranted.