The majority of HIV-1 strains enter CD4+ T cells using the CCR5 and/or CXCR4 co-receptor. However, we recently identified a transmitted/founder (T/F) virus (ZP6248) that efficiently used an alternative coreceptor GPR15, rather than commonly used CXCR4 and CCR5, to establish clinical infection. To understand which regions in the env gene were critical for the atypical coreceptor usage, we generated a set of V3 mutants and determined their infectivity in GHOST cells that expressed different coreceptors. When the variable loop 3 (V3) in YU2 was replaced with the ZP6248 V3 (YU2.6248V3), the chimera YU2.6248V3 infected GPR15+ cells but not CCR5+ cells. To determine which amino acids in V3 was responsible for this phenotype change, each of the eight amino acids that differed from the subtype B consensus V3 was substituted with alanine. The G306A and S322A mutations significantly reduced the replication capacity of YU2.6248V3 in GPR15+ cells, while all other alanine substitutions at positions 307, 314, 315, 316, 317 and 318 completely abrogated the infectivity of YU2.6248V3 in GPR15+ cells. The E314A mutation, as the E314G mutation reported before, also rendered the YU2.6248V3 infectious in CCR5+ cells, while none of other alanine mutants could infect CCR5+ cells. These results demonstrated that amino acids in ZP6248 V3 might form a unique conformation that was critical for the interaction with GPR15 while the amino acids at position 314 in the V3 crown of ZP6248 played a key role in interaction with both CCR5 and GPR15. The unique phenotypes of ZP6248 can serve as a model to understand how HIV-1 explores the diverse coreceptor reservoir through novel genetic variants to establish clinical infection.
Mucin 1 (MUC1) is a tumor-associated antigen that is overexpressed in several adenocarcinomas. However, clinical trials with MUC1 showed that MUC1 is a relatively poor immunogen in humans. In view of the low immunogenicity of this protein vaccine, we designed a method based on an immunoadjuvant and immunization strategy to enhance the cellular immune response to this protein vaccine. DDA/MPL has been evaluated as an adjuvant to induce strong immunity for the tuberculosis vaccine. However, its adjuvant role combined with the vaccine targeting MUC1 in malignant carcinomas has not previously been reported. Our previous study showed that adenovirus prime protein boost vaccination could significantly enhance the cellular immunity and antitumor efficacy. In our study, we used MUC1 VNTRs as the target of cancer vaccine and DDA/MPL as the adjuvant to enhancing the cellular immunity of recombinant MUC1 protein vaccine, and an AD-9M adenoviral vector prime-recombinant protein and DDA/MPL boost (designated MUC-1 VPP vaccine) strategy was studied to enhance the antitumor efficacy. The results demonstrated that antigen-specific IFN-?-secreting T cells were increased by 2-fold, and cytotoxic T lymphocytes (CTLs) were induced effectively when the protein vaccine was combined with the DDA/MPL adjuvant. Moreover, the vaccination induced nearly 60% inhibition of the growth of B16 melanoma in mice and prolonged the survival of tumor-bearing mice. The inhibition was correlated with the specific immune responses induced by the MUC1 VPP vaccine. The data suggested that DDA/MPL-adjuvant MUC-1 VPP vaccine may be developed into effective tumor vaccines for melanomas and possibly for other tumors expressing MUC1 protein.
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