Immune responses induced in HHD mice by multiepitope HIV vaccine based on cryptic epitope modification.

CD8+ T cells play an important role in early HIV infection. However, HIV has the capacity to avoid specific CTL responses due to a high rate of mutation under selection pressure. Although the HIV proteins, gag and pol, are relatively conserved, these sequences generate low-affinity MHC-associated epitopes that are poorly immunogenic. Here, we applied an approach that enhanced the immunogenicity of low-affinity HLA-A2.1-binding peptides. The first position with tyrosine (P1Y) substitution enhanced the affinity of HLA-A2.1-associated peptides without altering their antigenic specificity. More importantly, P1Y variants efficiently stimulated in vivo native peptide-specific CTL that also recognized the corresponding naturally processed epitope. The potential to generate CTL against any low-affinity HLA-A2.1-associated peptide provides us with the necessary technique for identification of virus cryptic epitopes for development of peptide-based immunotherapy. Therefore, identification and modification of the cryptic epitopes of gal and pol provides promising candidates for HIV immunotherapy dependent upon efficient presentation by virus cells. Furthermore, this may be a breakthrough that overcomes the obstacle of immune escape caused by high rates of mutation. In this study, bioinformatics analysis was used to predict six low-affinity cryptic HIV gag and pol epitopes presented by HLA-A*0201. A HIV compound multi-CTL epitope gene was constructed comprising the gene encoding the modified cryptic epitope and the HIV p24 antigen, which induced a strong CD8+ T cell immune response regardless of the mutation. This approach represents a novel strategy for the development of safe and effective HIV prophylactic and therapeutic vaccines.

[Immunogenicities and comparison of DNA vaccines encoding pol genes derived from B`/C and A/E recombinant HIV-1 strains].

OBJECTIVE: To construct and compare the immunogenicities of DNA vaccines expressing pol genes derived from B`/C and A/E recombinant subtypes of HIV-1 in China. METHODS: Two DNA vaccines were constructed by inserting the codon optimized pol genes derived from B'/C and A/E subtypes of HIV-1 into mammalian expression vector pSV1.0. In vitro expression efficiencies of the two DNA vaccines were determined by Western blotting and their immunogenicities were compared by i.m. immunizing female BALB/c mice. After immunization, mice splenocytes were isolated sterilely and IFN-γ based enzyme linked immunospot assay (ELISPOT) was employed to read out the specific T cell immunity. RESULTS: The constructed DNA vaccines were validated by restriction enzyme digestion and DNA sequencing. Western blotting result showed both of the two DNA vaccines could be expressed at appreciable levels in vitro. Under the stimulation of Consensus B Pol peptide pools, specific T cell frequency elicited by pSVAE-Pol was (636±178) SFCs/10(6) splenocytes; specific T cell frequency elicited by pSVCN-Pol was (468±265)SFCs/10(6) splenocytes (P=0.412). Under the stimulation of HIV-1 AE2f Pol peptide pools, specific T cell frequency elicited by pSVAE-Pol was (1378±611) SFCs/10(6) splenocytes; specific T cell frequency elicited by pSVCN-Pol was (713±61) SFCs/10(6) splenocytes (P=0.134). Further analysis suggested pSVAE-Pol induced specific T cell responses mainly focused on Pol 1 peptide pool, while, in addition to induce Pol 1 specific T cell responses, pSVCN-Pol could also elicit T cell responses against consensus B Pol 2 peptide pool. CONCLUSION: Although pSVAE-Pol was more immunogenic, pSVCN-Pol could induce T cell responses against broader epitope spectrum. Rational vaccine design may need combine them together.

HIV type 1 subtypes based on the pol gene and drug resistance mutations among antiretroviral-naive patients from Guangxi, Southern China.

We investigated the distribution of HIV-1 subtypes and the prevalence of HIV-1 drug resistance among HIV-infected antiretroviral-naive patients in Guangxi, southern China. A total of 144 subjects from Liuzhou or Nanning, two cities having the most HIV-infected cases in Guangxi, were enrolled. HIV-1 pol fragments were amplified and sequenced from plasma of all patients. In all, 124 sequences were obtained, with 67 from Liuzhou and 57 from Nanning. All sequences were subtyped by phylogenetic analysis and analyzed for antiretroviral resistance using the HIVdb program. Our data showed that the sequences from Liuzhou were subtyped as CRF01_AE (77.6%), CRF07_BC(20.9%), and BC (1.5%), respectively, and the sequences from Nanning as CRF01_AE (78.9%), CRF08_BC (15.8%), B (3.5%), and C (1.8%), respectively. Of the sequences 11.9% from Liuzhou and 28.1% from Nanning harbored drug resistance-associated mutations, but there were only two sequences with mutations associated with significantly reduced phenotypic susceptibility to antiretroviral drugs.

Infectivity and vaccination efficacy studies in animal models of HBV S and pol gene mutants.

Infectious HBV wild-type and mutant clones were produced in vitro with three mutations in pol (rtV173L plus rtL180M plus rtM204V) or with a single mutation in the S gene (sG145R) and inoculated in treatment-naive chimpanzees. Intravenous inoculation of these mutants in chimpanzees resulted in HBV infection; the pol mutations remained stable, whereas the sG145R mutation reverted to wild type during viraemia. Additional hepatitis B vaccine efficacy studies conducted in chimpanzees showed lack of sterilizing immunity against the pol mutant. Whether such mutants can transmit to and infect vaccinated humans requires further investigation.

Recombinant vaccinia DIs expressing simian immunodeficiency virus gag and pol in mammalian cells induces efficient cellular immunity as a safe immunodeficiency virus vaccine candidate.

A highly attenuated vaccinia virus substrain of Dairen-I (DIs) shows promise as a candidate vector for eliciting positive immunity against immune deficiency virus. DIs was randomly obtained by serial 1-day egg passages of a chorioarantoic membrane-adapted Dairen strain (DIE), resulting in substantial genomic deletion, including various genes regulating the virus-host-range. To investigate the impact of that deletion and of the subsequent insertion of a foreign gene into that region of DIs on the ability of the DIs recombinant to induce antigen-specific immunity, we generated a recombinant vaccinia DIs expressing fulllength gag and pol genes of simian immunodeficiency virus (SIV) (rDIsSIV gag/pol) and studied the biological and immunological characteristics of the recombinant natural mutant. The rDIsSIV gag/pol developed a tiny plaque on the chick embryo fibroblast (CEF). Viral particles of rDIsSIV gag/pol as well as SIV Gag-like particles were electromicroscopically detected in the cytoplasm. Interestingly, the recombinant DIs strain grows well in CEF cells but not in mammalian cells. While rDIsSIV gag/pol produces SIV proteins in mammalian HeLa and CV-1 cells, recombinant modified vaccinia Ankara strain (MVA) expressing SIV gag and pol genes (MVA/SIV239 gag/pol) clearly replicates in HeLa and CV-1 cell lines under synchronized growth conditions and produces the SIV protein in all cell lines. Moreover, intradermal administration of rDIsSIV gag/pol or of MVA/SIV239 gag/pol elicited similar levels of IFN-gamma spot-forming cells specific for SIV Gag. If the non-productive infection characteristically induced by recombinant DIs is sufficient to trigger immune induction, as we believe it is, then a human immunodeficiency virus vaccine employing the DIs recombinant would have the twin advantages of being both effective and safe.

Persistence of primary drug resistance among recently HIV-1 infected adults.

OBJECTIVES: Primary, or transmitted, drug resistance is common among treatment naive patients recently infected with HIV-1, and impairs response to anti-retroviral therapy. We previously observed that patients with secondary resistance (developed in response to anti-retroviral treatment) who chose to stop an anti-retroviral regimen experience rapid overgrowth of drug resistant viruses by wild-type virus of higher pol replication capacity. We sought to determine if primary drug resistance would be lost at a rapid rate, and viral pol replication capacity would increase, in the absence of treatment. METHODS: We tracked drug resistance phenotype, genotype, viral pol replication capacity (single cycle recombinant assay incorporating a segment of the patient pol gene [pol RC]), plasma HIV-1 RNA, and CD4 T cell counts in the absence of treatment among patients in early HIV-1 infection. RESULTS: Six of 22 patients had evidence of primary drug resistance to at least one class of drug; three resistant to protease inhibitors, three resistant to non-nucleoside reverse transcriptase inhibitors, and four resistant to nucleoside reverse transcriptase inhibitors. All six patients maintained evidence of drug resistance for the period of observation. Among patients with baseline primary drug resistance pol RC did not increase over time. CONCLUSION: The selection environment of early infection is determined by immune pressure, and stochastic events, not viral pol replication capacity. In contrast to secondary resistant infections that are rapidly overgrown when therapy is stopped, primary drug resistance persists over time. Surveillance and clinical detection of primary resistance is feasible in the first year of infection.

Vaccine-induced immune responses in rodents and nonhuman primates by use of a humanized human immunodeficiency virus type 1 pol gene.

A synthetic gene consisting of the reverse transcriptase (RT) and integrase (IN) domains of human immunodeficiency virus type 1 (HIV-1) pol was constructed using codons most frequently used in humans. The humanized pol gave dramatically improved levels of Rev-independent, in vitro protein production in mammalian cells and elicited much stronger cellular immunity in rodents than did virus-derived gene. Specifically, BALB/c mice were immunized with plasmids and/or recombinant vaccinia virus constructs expressing the synthetic gene. High frequencies of Pol-specific T lymphocytes were detected in these animals by the gamma interferon enzyme-linked immunospot assay against pools of short overlapping peptides. Characterization of the stimulatory peptides from these pools indicates that the optimized gene constructs are able to effectively activate both CD4+ and CD8+ T cells. Immunization of rhesus macaques with DNA vaccines expressing the humanized pol coupled to a human tissue plasminogen activator leader sequence led to pronounced in vitro cytotoxic T-lymphocyte killing activities and enhanced levels of circulating Pol-specific T cells, comparable to those observed in HIV-1-infected human subjects. Thus, optimizing the immunogenic properties of HIV-1 Pol at the level of the gene sequence validates it as an antigen and provides an important step toward the construction of a potent pol-based HIV-1 vaccine component.

Human immunodeficiency virus type 1-specific immunity after genetic immunization is enhanced by modification of Gag and Pol expression.

Immunity to human immunodeficiency virus virion-like structures or a polyprotein has been examined after DNA immunization with Rev-independent expression vectors. A Gag-Pol fusion protein stimulated cytotoxic T lymphocyte and antibody responses to Gag and Pol, while a Gag-Pol pseudoparticle did not elicit substantial Pol responses. This fusion protein may be useful for AIDS vaccines.

Therapeutic immunization of HIV-infected chimpanzees using HIV-1 plasmid antigens and interleukin-12 expressing plasmids.

OBJECTIVE: To assess HIV-1 DNA vaccination and co-immunization with interleukin (IL)-12 and IL-10 as immunotherapy in the HIV-1 infected chimpanzee model system. METHODS: Four chimpanzees that were infected with HIV-1-IIIB for longer than 4 years and remained symptom free were immunized with HIV-1 plasmid vaccines. Two chimpanzees were immunized with DNA plasmids that encoded env/rev, gag/pol along with a plasmid that encoded both chains of human IL-12. A third animal was immunized with HIV-1 DNA vaccine constructs and co-immunized with an IL-10 expressing plasmid. Finally a control animal received the HIV-1 DNA vaccine constructs alone. RESULTS: There was no evidence of systemic toxicity associated with the administration of the DNA vaccines or the cytokine-expressing plasmids. We observed that the IL-12/HIV-1 DNA vaccinated animals had enhanced proliferative responses to multiple HIV-1 antigens at multiple time points. The animal that was co-immunized with HIV-1 and IL-10 did not have any changes in the proliferative responses. Finally, the control chimpanzee demonstrated moderate increases in the proliferative responses to HIV-1 antigens. The animal that received HIV-1 vaccines alone and the animals co-immunized with IL-12 all had declines in viral load over the course of the study, however, the decrease in viral loads were transient in all animals. CONCLUSION: Immunization of HIV-1 infected chimpanzees with DNA based vaccines containing the env, gag and pol genes can transiently boost the env specific proliferative responses. Co-administration of IL-12 expressing plasmids further leads to transient boosting of the proliferative response to the core protein, p24 as well. However, at these doses the impact on viral load is minimal.