Microparticles in MF59, a potent adjuvant combination for a recombinant protein vaccine against HIV-1

Novel adjuvant formulations involving PLG microparticles with entrapped recombinant protein antigens (env gp120 and p24 gag) from human immunodeficiency virus type-1 (HIV-1), dispersed in the emulsion adjuvant MF59 were evaluated as potential HIV-1 vaccine candidates in mice and baboons. In mice, the adjuvant combination induced significantly enhanced antibody responses in comparison to either adjuvant used alone. In addition, the polylactide co-glycolide polymer (PLG) microparticles and MF59 combination induced CTL activity against HIV-1 p24 gag. In baboons, the adjuvant combination induced significantly enhanced antibody titers after a single dose of gp120, but the responses were comparable to gp120 in MF59 alone after boosting. Both MF59+gp120 alone and PLG/gp120 in MF59 induced neutralizing antibodies against a T cell line-adapted (TCLA) strain and a primary isolate of HIV-1. In contrast to the observations with gp120, immunization in baboons with PLG/p24 in MF59 induced significantly enhanced antibody responses after boosting, in comparison to immunization with MF59 alone + p24.     

Immunization with an HIV-1 immunogen induces CD4+ and CD8+ HIV-1-specific polyfunctional responses in patients with chronic HIV-1 infection receiving antiretroviral therapy

Development of polyfunctional T lymphocyte responses is critical in the immunological response against HIV-1. Fifty-four HIV-1 infected patients receiving antiretroviral treatment (ART) and immunization with an HIV-1 immunogen or placebo, periodically every 3 months throughout a period of 36 months, were evaluated for the purposes of analysing the development of HIV-1-specific CD4⁺ and CD8⁺ responses. A significant increase of proliferating and IFN-γ producing CD8⁺ HIV-1-specific T cells, of HIV-1-specific precursor frequencies for CD8⁺ and for CD4⁺ T cells and of Gag/pol-specific memory CTL precursors (CTLp) was observed in the immunogen group in comparison to placebo. IL-2 intracellular expression and IFN-γ and TNF- co-expression in HIV-1-specific CD8⁺ T cells were also substantially increased in the immunized group. A negative correlation between viral load and CD3⁺CD4⁺CFSE^low HIV-1-specific lymphoproliferative response and frequency of Gag/pol-specific CTLp was solely observed in the HIV-1 immunogen group. Long-term immunization in patients receiving ART helps to develop HIV-1-specific polyfunctional T cell responses.     

Strong and persistent CD4 T-cell response in healthy adults immunized with a candidate HIV-1 vaccine containing gp120, Nef and Tat antigens formulated in three Adjuvant Systems

This randomized double-blind study aimed to determine the safety and immunogenicity of a gp120/NefTat candidate human immunodeficiency virus type 1 (HIV-1) vaccine formulated with one of three different Adjuvant Systems (AS02_A, AS02_V and AS01_B) in healthy HIV-seronegative adults. All vaccine formulations induced strong HIV-specific CD4⁺ T-cell responses characterized by high lymphoproliferative capacity and IL-2 production that were still detectable 18 months after last immunization, with strongest responses seen in the AS01_B group. Broad coverage was demonstrated against gp120, and to a lesser extent Nef, derived from the most common circulating clades (B, C and circulating recombinant form [CRF]-01). All vaccine formulations exhibited acceptable safety and reactogenicity profiles. The demonstration of superior CD4⁺ T-cell induction by AS01_B provides important guidance for future HIV vaccine development.     

Delayed progression of murine AIDS in C57BL/6 mice pre-immunized with a highly antigenic 10-mer peptide encoded by the murine AIDS defective virus gag p12 gene

C57BL/6 (B6) mice were immunized with a highly antigenic 10-mer peptide (P12-10), which is encoded by the murine AIDS (MAIDS) defective virus gag p12 gene, emulsified in incomplete Freund's adjuvant (ICFA). One week later, the mice were inoculated with the MAIDS virus to see if the immunization affects progression of MAIDS. It was demonstrated that the immunization significantly delayed progression of MAIDS, although it failed to induce appreciable cytotoxic T lymphocyte (CTL) responses against the P12-10 antigen. In constrast, immunization of B6 mice with the P12-10 coupled with liposome induced substantial CTL responses but failed to protect the mice against MAIDS development. This segregation between CTL activity and in vivo protection efficacy might be worth considering when we exploit vaccines for augmenting cellular immunity mediated by CD8⁺ T cells.     

Long-term persistence of vaccination and HAART to human immunodeficiency virus (HIV)

The aim of this study was to monitor the immune responses in HIV-infected patients previously immunized with gp160 or DNA vaccines to analyze whether the introduction of highly active antiretroviral treatment (HAART) would affect the persistence of immunity. The immune responses were evaluated in patients who had participated in randomized trials of therapeutic vaccination. Immunization in conjunction with antiretroviral therapy was effective in inducing HIV-specific T-cell responses. Therapeutic immunizations with recombinant gp160 had a modest effect on CD4-cell counts, the treatment alone lead to a transient clinical benefit in the form of an improved survival after two years of immunization. Immunizations with HIV DNA during HAART treatment permitted persistence or development of innate (NK), CD4+ and/or CD8+ immune responses. HIV specific T-helper cell responses induced by immunization with gp160 were maintained at high levels up to 7 years after the last injection. Cells with HIV-specific interferon-gamma (IFN-gamma) production were retained or increased in long-term HAART treated patients. The impact of a single structured therapy interruption (STI) was analyzed in a small group of patients showing no obvious increase or decrease in the HIV-specific immune response during or after STI. The possibility to induce very long-term strong and persistent immune responses in HIV-infected individuals raises hopes that vaccination preceding therapy interruption might prolong the symptom-free period without HAART.     

DNA vaccine expressing HIV-1 gp120/immunoglobulin fusion protein enhances cellular immunity

In this study, we explored the possibility of augmenting human immunodeficiency virus (HIV) gp120-specific cell-mediated immune responses in mice by means of a DNA vaccine encoding a mouse Ig Fcγ2a fragment fused with gp120 (gp120-Ig, Ig-gp120). Western blotting analysis revealed that the HIV gp120 protein expression efficiency was higher in cells transfected with the gp120-Ig-coding plasmid (pGp120Ig) than in those transfected with the gp120 and Ig-gp120 expression plasmids (pGp120 and pIgGp120, respectively). pGp120Ig elicited more HIV-specific CD8 T cells and effector memory CD8 T cells than pGp120 in immunized mice. Furthermore, pGp120Ig significantly reduced the viral load after challenge with an HIV Env gp160-expressing vaccinia virus. These results demonstrate that covalent antigen modification with an Ig sequence can modulate antigen-specific cellular immune responses. The approach may be useful for vaccine development.     

新报告HIV感染者/AIDS基线病毒载量水平与CD4+/CD8+比值分析

目的 分析江西省2015—2019年新报告艾滋病病毒(HIV)感染者/艾滋病(AIDS)病人(简称HIV感染者/AIDS)的基线病毒载量水平与其细胞免疫水平之间的关系。方法 对2015—2019年在江西省疾病预防控制中心确证的HIV感染者/AIDS进行基线CD4⁺T淋巴细胞、CD8⁺T淋巴细胞和病毒载量检测,用SPSS软件对数据进行相关性分析和二元logistic回归分析。结果 新报告HIV感染者/AIDS 513例,CD4⁺T淋巴细胞计数中位数为209个/μl,CD4⁺/CD8⁺比值中位数为0.233,病毒载量中位数为5.06 log₁₀ 拷贝/ml。病毒载量水平与CD4⁺T淋巴细胞、CD4⁺/CD8⁺比值总体呈显著负相关; CD4⁺/CD8⁺比值<0.20 的HIV感染者/AIDS其病毒载量≥10⁵拷贝/ml的风险为CD4⁺/CD8⁺比值≥0.20的3.775倍。结论 江西省新报告HIV感染者/AIDS中高病毒载量比例高,中位病毒载量高,CD4⁺/CD8⁺比值<0.2是高病毒载量的预测因素。应进一步加强扩大检测人群和比例,尽早发现感染者并进行抗病毒治疗,提高患者期望寿命。     

Immunization strategies to augment oral vaccination with DNA and viral vectors expressing HIV envelope glycoprotein

Induction of mucosal immunity to the human immunodeficiency virus (HIV) envelope (env; gp160) glycoprotein has been demonstrated with orally administered recombinant vaccinia virus (rVV) vectors and poly(DL-lactide-co-glycolide) (PLG)-encapsulated plasmid DNA expressing gp160. In this study, we investigated the effect of an oral DNA-prime/rVV-boost vaccine regimen in conjunction with adjuvants on the level of gp160-specific cellular and humoral responses in BALB/c mice. We demonstrated that DNA priming followed by a booster with rVV expressing gp160 (vPE16) significantly augmented env-specific immunity in systemic and mucosal tissues of the immunized mice. Association of vPE16 with liposomes and coadministration of liposome-associated beta-glucan lentinan or IL-2/Ig-encoded plasmid DNA-encapsulated in PLG microparticles triggered the optimal cell-mediated immune (CMI) responses. Lentinan was found to increase env-specific type 1 cytokine production and cytotoxic T-lymphocyte (CTL) activities but had no effect on humoral responses. On the other hand, IL-2/Ig-mediated increases in both type 1 and 2 activities were associated with higher levels of env-specific CTL and antibody responses. Results of these studies demonstrated the effectiveness of oral vaccines with DNA and rVV vectors in conjunction with immunomodulators in inducing specific immune responses in systemic and mucosal tissues.     

Durable HIV-1 antibody and T-cell responses elicited by an adjuvanted multi-protein recombinant vaccine in uninfected human volunteers

BACKGROUND: Use of the recombinant proteins NefTat and gp120(W61D) formulated with the AS02A adjuvant system was previously shown to protect against AIDS in a rhesus macaque SHIV animal model system. METHODS: Eighty-four HIV uninfected human participants were vaccinated intramuscularly at 0, 1, and 3 months and evaluated for safety. Immune responses were analyzed for the presence of vaccine-induced antibody and T lymphocyte responses. RESULTS: The vaccines were safe and well tolerated at all doses. Nef-, Tat-, and gp120-specific binding antibodies were induced in all individuals that received the respective antigen, lasting up to 9 months after the final immunization. Antibodies able to neutralize the T-cell laboratory-adapted strain of HIV-1(W61D) were detected in the majority of vacinees, but did not neutralize primary isolates. Envelope-specific antibody-dependent cell cytoxicity was detected in most of the individuals receiving gp120. Robust and persistent HIV-specific lymphoproliferative responses were detected against all subunit proteins in the majority of immunized participants. As expected, HIV-specific CD8 T-cell responses were not detected. CONCLUSIONS: Despite the lack of primary isolate neutralizing antibody induction, the observed high frequency and magnitude of other immune responses warrant further work with this vaccine or vaccine components.     

Oral immunization of rhesus macaques with adenoviral HIV vaccines using enteric-coated capsules

Targeted delivery of vaccine candidates to the gastrointestinal (GI) tract holds potential for mucosal immunization, particularly against mucosal pathogens like the human immunodeficiency virus (HIV). Among the different strategies for achieving targeted release in the GI tract, namely the small intestine, pH sensitive enteric coating polymers have been shown to protect solid oral dosage forms from the harsh digestive environment of the stomach and dissolve relatively rapidly in the small intestine by taking advantage of the luminal pH gradient. We developed an enteric polymethacrylate formulation for coating hydroxy-propyl-methyl-cellulose (HPMC) capsules containing lyophilized Adenoviral type 5 (Ad5) vectors expressing HIV-1 gag and a string of six highly-conserved HIV-1 envelope peptides representing broadly cross-reactive CD4⁺ and CD8⁺ T cell epitopes. Oral immunization of rhesus macaques with these capsules primed antigen-specific mucosal and systemic immune responses and subsequent intranasal delivery of the envelope peptide cocktail using a mutant cholera toxin adjuvant boosted cellular immune responses including, antigen-specific intracellular IFN-γ-producing CD4⁺ and CD8⁺ effector memory T cells in the intestine. These results suggest that the combination of oral adenoviral vector priming followed by intranasal protein/peptide boosting may be an effective mucosal HIV vaccination strategy for targeting viral antigens to the GI tract and priming systemic and mucosal immunity.     

Gp96 SIV Ig immunization induces potent polyepitope specific, multifunctional memory responses in rectal and vaginal mucosa

The ER-resident chaperone gp96, when released by cell lysis, induces an immunogenic chemokine signature and causes innate immune activation of DC and NK cells. Here we show that intraperitoneal immunization with a genetically engineered, secreted form of gp96, gp96-Ig chaperoning SIV antigens, induces high levels of antigen specific CD8 CTL in the rectal and vaginal mucosa of Rhesus macaques. The frequency of SIV Gag- and SIV Tat-tetramer positive CD8 CTL in the intestinal mucosa reached 30-50% after the third immunization. Tetramer positive CD8 CTL expressed appropriate functional (granzyme B) and migration markers (CD103). The polyepitope specificity of the mucosal CD8 and CD4 response is evident from a strong, multifunctional cytokine response upon stimulation with peptides covering the gag, tat and env proteins. Induction of powerful mucosal effector CD8 CTL responses by cell-based gp96^SIV-Ig immunization may provide a pathway to the development of safe and effective SIV/HIV vaccines.     

The HIV-1 chimeric protein CR3 expressed by poxviral vectors induces a diverse CD8+ T cell response in mice and is antigenic for PBMCs from HIV+ patients

Recombinant avipoxvirus vectors are attractive for vaccination against human immunodeficiency virus type 1 (HIV-1), where induction of a cytotoxic CD8(+) T cell (CTL) response seems to be an important component of protective immunity. We expressed the chimeric protein CR3, composed by CTL epitopes rich regions from, RT, Gag and Nef and conserved Th cell epitopes from gp120, gp41 and Vpr of HIV-1 in a fowlpox virus (FWPV) vector (FPCR3), and used this vector to induce HIV-specific CTL responses in mice. Mice immunised twice intraperitoneally with FPCR3, developed a CD8(+) T cell response measured as production of IFN-gamma by splenocytes in response to stimulation with P815 cells infected with recombinant vaccinia viruses (rVV) expressing CR3, Gag and Nef. The number of IFN-gamma secreting cells was markedly higher when a P815 cell line constitutively expressing CR3 was used as target cells for Enzyme-linked-immunospot (ELISPOT). CR3 epitopes were also specifically recognised by human PBMCs from three HIV(+) patients with different haplotypes. These results confirm the potential of FWPV vectors expressing these novel HIV-1 chimeric proteins to induce a simultaneous CD8(+) T cell response against conserved viral targets and early expressed regulatory proteins.     

探讨在细菌性感染及病毒性感染疾病中淋巴细胞亚群的表达水平及意义

目的探讨不同感染性疾病患者外周血中T、CD3⁺CD4⁺T、CD3⁺CD8⁺T 、CD4^-CD8^-T、CD4⁺CD8⁺T、B、NK、CD4⁺Treg、CD8⁺Treg、CTL及CD28^-CTL等淋巴细胞亚群的表达水平,为不同感染性疾病的辅助诊断、免疫监测以及临床治疗提供依据。 方法选取本院2017年10月-2018年2月诊治的不同类型感染性疾病86例,其中败血症26例,HBV感染30例,EBV感染30例,对照组30例;用流式细胞术检测各组淋巴细胞亚群表达水平。 结果与对照组相比,HBV组、EBV组、败血症组患者的外周血CD3⁺CD4⁺T、CD3⁺CD8⁺T 、CD4⁺CD8⁺T、B、NK、CD4⁺Treg、CTL及CD28^-CTL等淋巴细胞亚群的表达水平差异具有统计学意义(P<0.05),HBV组中CD3⁺CD4⁺T和CTL升高,NK和CD28^-CTL降低;EBV组中CD3⁺CD8⁺T、CD4⁺Treg和CTL升高,CD3⁺CD4⁺T和NK降低;败血症组中B和CTL升高,CD4^-CD8^-T、NK和CD28^-CTL降低。与败血症和HBV组相比,EBV组的CD3⁺CD4⁺T表达降低,CD3⁺CD8⁺T和CTL表达升高,差异具有统计学意义(P<0.05);与HBV组相比,败血症组CD4^-CD8^-T表达降低,与EBV组相比,败血症组B表达升高,HBV组CD28^-CTL降低,差异具有统计学意义(P<0.05)。另外,比较发现HBV组、EBV组、败血症组三组患者的外周血中均表现为CTL表达升高,NK表达降低。 结论不同感染性疾病患者外周血中CD3⁺CD4⁺T、CD3⁺CD8⁺T 、CD4^-CD8^-T 、B、NK、CD4⁺Treg、CTL及CD28^-CTL等淋巴细胞亚群的表达水平存在差异,通过分析其表达水平的变化可为不同感染性疾病的辅助诊断、免疫监测以及临床治疗提供依据。     

Gene gun DNA vaccination with Rev-independent synthetic HIV-1 gp160 envelope gene using mammalian codons.

DNA immunization with HIV envelope plasmids induce only moderate levels of specific antibodies which may in part be due to limitations in expression influenced by a species-specific and biased HIV codon usage. We compared antibody levels, Th1/Th2 type and CTL responses induced by synthetic genes encoding membrane bound gp160 versus secreted gp120 using optimized codons and the efficient gene gun immunization method. The in vitro expression of syn.gp160 as gp120 + gp41 was Rev independent and much higher than a classical wt.gp160 plasmid. Mice immunized with syn.gp160 and wt.gp160 generated low and inconsistent ELISA antibody titres whereas the secreted gp120 consistently induced faster seroconversion and higher antibody titres. Due to a higher C + G content the numbers of putative CpG immune (Th1) stimulatory motifs were highest in the synthetic gp160 gene. However, both synthetic genes induced an equally strong and more pronounced Th2 response with higher IgG1/IgG2a and IFNgamma/IL-4 ratios than the wt.gp160 gene. As for induction of CTL, synthetic genes induced a somewhat earlier response but did not offer any advantage over wild type genes at a later time point. Thus, optimizing codon usage has the advantage of rendering the structural HIV genes Rev independent. For induction of antibodies the level of expression, while important, seems less critical than optimal contact with antigen presenting cells at locations reached by the secreted gp120 protein. A proposed Th1 adjuvant effect of the higher numbers of CpG motifs in the synthetic genes was not seen using gene gun immunization which may be due to the low amount of DNA used.     

Effectiveness of intranasal immunization with HIV-gp160 and an HIV-1 env CTL epitope peptide (E7) in combination with the mucosal adjuvant LT(R192G)

LT(R192G) is a novel mucosal adjuvant that induces protective immunity when co-administered with certain whole inactivated bacteria or viruses or with subunits of relevant virulence determinants from these pathogens. LT(R192G) stimulates antigen-specific humoral and cellular immune responses, both systemically and in mucosal compartments, and is safe and nontoxic at adjuvant effective doses. Intranasal (IN) immunization of mice with LT(R192G) in conjunction with oligomeric HIV-1 gp160 elevates antigen-specific systemic and mucosal IgG and IgA production and Th1- and Th2-type cytokine responses. Isotype characterization of induced IgG reveals that gp160 alone fails to stimulate IgG2a responses in the absence of adjuvant. Both IgG1 and IgG2a are induced by immunization in the presence of LT(R192G). Additionally, intranasal immunization with a 15-amino acid peptide corresponding to an HIV-1 Env CTL determinant and LT(R192G) induces systemic, peptide-specific CTL activity and Th1 and Th2 cytokine responses that are absent when the adjuvant is excluded from the immunizations. These studies show that LT(R192G) quantitatively and qualitatively enhances cellular and humoral HIV-specific immune responses and that this adjuvant may offer significant advantages toward vaccine development against HIV.     

Safety and immunogenicity of an HIV-1 gp120-CD4 chimeric subunit vaccine in a phase 1a randomized controlled trial

A major challenge for HIV vaccine development is to raise anti-envelope antibodies capable of recognizing and neutralizing diverse strains of HIV-1. Accordingly, a full length single chain (FLSC) of gp120-CD4 chimeric vaccine construct was designed to present a highly conserved CD4-induced (CD4i) HIV-1 envelope structure that elicits cross-reactive anti-envelope humoral responses and protective immunity in animal models of HIV infection. IHV01 is the FLSC formulated in aluminum phosphate adjuvant. We enrolled 65 healthy adult volunteers in this first-in-human phase 1a randomized, double-blind, placebo-controlled study with three dose-escalating cohorts (75 µg, 150 µg, and 300 µg doses). Intramuscular injections were given on weeks 0, 4, 8, and 24. Participants were followed for an additional 24 weeks after the last immunization. The overall incidence of adverse events (AEs) was not significantly different between vaccinees and controls. The majority (89%) of vaccine-related AE were mild. The most common vaccine-related adverse event was injection site pain. There were no vaccine-related serious AE, discontinuation due to AE, intercurrent HIV infection, or significant decreases in CD4 count. By the final vaccination, all vaccine recipients developed antibodies against IHV01 and demonstrated anti-CD4i epitope antibodies. The elicited antibodies reacted with CD4 non-liganded Env antigens from diverse HIV-1 strains. Antibody-dependent cell-mediated cytotoxicity against heterologous infected cells or gp120 bound to CD4+ cells was evident in all cohorts as were anti-gp120 T-cell responses. IHV01 vaccine was safe, well tolerated, and immunogenic at all doses tested. The vaccine raised broadly reactive humoral responses against conserved CD4i epitopes on gp120 that mediates antiviral functions.     

Polylactide-co-glycolide (PLG) microparticles modify the immune response to DNA vaccination

Priming with the major surface glycoprotein G of respiratory syncytial virus (RSV) expressed by recombinant vaccinia leads to strong Th2 responses and lung eosinophilia during viral challenge. We now show that DNA vaccination in BALB/c mice with plasmids encoding G attenuated RSV replication but also enhanced disease with lung eosinophilia and increased IL-4/5 production. However, formulating the DNA with PLG microparticles reduced the severity of disease during RSV challenge without significantly lessening protection against viral replication. PLG formulation greatly reduced lung eosinophilia and prevented the induction of IL-4 and IL-5 during challenge, accompanied by a less marked CD4+ T cell response and a restoration of the CD8+ T cell recruitment seen during infection of non-vaccinated animals. After RSV challenge, lung eosinophilia was enhanced and prolonged in mice vaccinated with DNA encoding a secreted form of G; this effect was virtually prevented by PLG formulation. Therefore, PLG microparticulate formulation modifies the pattern of immune responses induced by DNA vaccination boosts CD8+ T cell priming and attenuates Th2 responses. We speculate that PLG microparticles affect antigen uptake and processing, thereby influencing the outcome of DNA vaccination.     

Cationic microparticles are a potent delivery system for a HCV DNA vaccine

We initially evaluated in mice the ability of naked DNA encoding intracellular forms of the E1E2 envelope proteins from HCV to induce antibody responses and compared the responses induced with the same plasmid adsorbed onto cationic poly (lactide co-glycolide) (PLG) microparticles. Although naked DNA was only able to induce detectable responses at the 100 microg dose level, making this approach impractical for evaluation in larger animals, PLG/DNA induced detectable responses at 10 microg. In addition, the PLG/DNA microparticles induced significantly enhanced responses to naked DNA when compared at the same dose level. Remarkably, PLG/DNA induced comparable responses to recombinant E1E2 protein adjuvanted with the emulsion MF59. Furthermore, PLG/DNA effectively primed for a booster response with protein immunization, while naked DNA did not. Therefore, PLG/DNA was selected for further evaluation in a non-human primate model. In a study in rhesus macaques, PLG/DNA induced seroconversion in 3/3 animals following three immunizations. Although the antibody responses appeared lower than those induced with recombinant protein adjuvanted with MF59, following a fourth dose, PLG/DNA and protein induced comparable responses. However, a single booster dose of recombinant protein administered to the animals previously immunized with PLG/DNA induced much higher responses. In addition, one of three animals immunized with PLG/DNA showed a cytotoxic T lymphocyte response in peripheral blood lymphocytes. In conclusion, cationic PLG microparticles with adsorbed HCV DNA generates potent immune responses.     

Induction of gp120-specific protective immune responses by genetic vaccination with linear polyethylenimine-plasmid complex

The induction of IFN-gamma-secreting CD8+ T cells and neutralizing antibodies to HIV-1 are both key requirements for prevention of viral transmission and clearance of pathogenic HIV. Although DNA vaccination has been shown to induce both humoral and cellular immune responses against HIV antigens, the magnitude of the immune responses has always been disappointing. In this report, we analyze the ability of polyethylenimine (PEI)-DNA complex expressing an HIV-glycoprotein 120 (gp120) antigen (PEI-pgp120) to induce systemic CD8+ T cell and humoral responses to the gp120 antigen. The administration of PEI-plasmid complex resulted in rapid elevation of serum levels of IL-12 and IFN-gamma. Furthermore, a single administration of PEI-pgp120 complex elicits a number of gp120-specific CD8+ T cells 20 times higher than that elicited by three intramuscular injections of naked DNA. Interestingly, we found that systemic vaccination with PEI-pgp120 induced protective immune responses against both systemic and mucosal challenges with a recombinant vaccinia virus expressing a gp120 antigen. The data also demonstrated that the depletion of macrophages with liposome-encapsulated clodronate completely abolished gp120-specific cellular response. Overall, our results showed that a single administration of PEI-pgp120 complexes, eliciting strong immune responses, is an effective vaccination approach to generate protection against systemic and mucosal viral infections.     

Recombinant influenza virus expressing HIV-1 p24 capsid protein induces mucosal HIV-specific CD8 T-cell responses

Influenza viruses are promising mucosal vaccine vectors for HIV but their use has been limited by difficulties in engineering the expression of large amounts of foreign protein. We developed recombinant influenza viruses incorporating the HIV-1 p24 gag capsid into the NS-segment of PR8 (H1N1) and X31 (H3N2) influenza viruses with the use of multiple 2A ribosomal skip sequences. Despite the insertion of a sizable HIV-1 gene into the influenza genome, recombinant viruses were readily rescued to high titers. Intracellular expression of p24 capsid was confirmed by in vitro infection assays. The recombinant influenza viruses were subsequently tested as mucosal vaccines in BALB/c mice. Recombinant viruses were attenuated and safe in immunized mice. Systemic and mucosal HIV-specific CD8 T-cell responses were elicited in mice that were immunized via intranasal route with a prime-boost regimen. Isolated HIV-specific CD8 T-cells displayed polyfunctional cytokine and degranulation profiles. Mice boosted via intravaginal route induced recall responses from the distal lung mucosa and developed heightened HIV-specific CD8 T-cell responses in the vaginal mucosa. These findings demonstrate the potential utility of recombinant influenza viruses as vaccines for mucosal immunity against HIV-1 infection.