Specific properties of shRNA-mediated CCR5 downregulation that enhance the inhibition of HIV-1 infection in combination with shRNA targeting HIV-1 rev.

Treatment with RNAi against HIV-1 transcripts efficiently inhibits viral replication but induces selection of escape mutants; therefore, the CCR5 coreceptor was suggested as an additional target. Blocking viral and host transcripts improved the antiviral effect. We have used short hairpin RNA (shRNA) targeting the human CCR5 (shCCR5) or the HIV-1 rev (shRev) transcripts to demonstrate distinctive properties of anti-CCR5 shRNA: shCCR5 induced more sustained protection than shRev; partial reduction in CCR5 expression substantially decreased HIV-1 infection, and shCCR5 performed better than shRev in the mixed shRNA-treated and untreated cultures. These observations indicate that CCR5 inhibitors should be conveniently included in HIV-1 gene silencing treatment schedules when only a certain cell fraction is protected to further reduce endogenous virus in a properly ART-treated HIV-1 infected individual.

HIV-1 Protease as DNA Immunogen against Drug Resistance in HIV-1 Infection: DNA Immunization with Drug Resistant HIV-1 Protease Protects Mice from Challenge with Protease-Expressing Cells.

DNA immunization with HIV-1 protease (PR) is advanced for immunotherapy of HIV-1 infection to reduce the number of infected cells producing drug-resistant virus. A consensus PR of the HIV-1 FSU_A strain was designed, expression-optimized, inactivated (D25N), and supplemented with drug resistance (DR) mutations M46I, I54V, and V82A common for FSU_A. PR variants with D25N/M46I/I54V (PR_Ai2mut) and with D25N/M46I/I54V/V82A (PR_Ai3mut) were cloned into the DNA vaccine vector pVAX1, and PR_Ai3mut, into a lentiviral vector for the transduction of murine mammary adenocarcinoma cells expressing luciferase 4T1luc2. BALB/c mice were DNA-immunized by intradermal injections of PR_Ai, PR_Ai2mut, PR_Ai3mut, vector pVAX1, or PBS with electroporation. All PR variants induced specific CD8+ T-cell responses revealed after splenocyte stimulation with PR-derived peptides. Splenocytes of mice DNA-immunized with PR_Ai and PR_Ai2mut were not activated by peptides carrying V82A, whereas splenocytes of PR_Ai3mut-immunized mice recognized both peptides with and without V82A mutation. Mutations M46I and I54V were immunologically silent. In the challenge study, DNA immunization with PR_Ai3mut protected mice from the outgrowth of subcutaneously implanted adenocarcinoma 4T1luc2 cells expressing PR_Ai3mut; a tumor was formed only in 1/10 implantation sites and no metastases were detected. Immunizations with other PR variants were not protective; all mice formed tumors and multiple metastasis in the lungs, liver, and spleen. CD8+ cells of PR_Ai3mut DNA-immunized mice exhibited strong IFN-γ/IL-2 responses against PR peptides, while the splenocytes of mice in other groups were nonresponsive. Thus, immunization with a DNA plasmid encoding inactive HIV-1 protease with DR mutations suppressed the growth and metastatic activity of tumor cells expressing PR identical to the one encoded by the immunogen. This demonstrates the capacity of T-cell response induced by DNA immunization to recognize single DR mutations, and supports the concept of the development of immunotherapies against drug resistance in HIV-1 infection. It also suggests that HIV-1-infected patients developing drug resistance may have a reduced natural immune response against DR HIV-1 mutations causing an immune escape.

Cellular Immune Response Induced by DNA Immunization of Mice with Drug Resistant Integrases of HIV-1 Clade A Offers Partial Protection against Growth and Metastatic Activity of Integrase-Expressing Adenocarcinoma Cells.

Therapeutic DNA-vaccination against drug-resistant HIV-1 may hinder emergence and spread of drug-resistant HIV-1, allowing for longer successful antiretroviral treatment (ART) up-to relief of ART. We designed DNA-vaccines against drug-resistant HIV-1 based on consensus clade A integrase (IN) resistant to raltegravir: IN_in_r1 (L74M/E92Q/V151I/N155H/G163R) or IN_in_r2 (E138K/G140S/Q148K) carrying D64V abrogating IN activity. INs, overexpressed in mammalian cells from synthetic genes, were assessed for stability, route of proteolytic degradation, and ability to induce oxidative stress. Both were found safe in immunotoxicity tests in mice, with no inherent carcinogenicity: their expression did not enhance tumorigenic or metastatic potential of adenocarcinoma 4T1 cells. DNA-immunization of mice with INs induced potent multicytokine T-cell response mainly against aa 209-239, and moderate IgG response cross-recognizing diverse IN variants. DNA-immunization with IN_in_r1 protected 60% of mice from challenge with 4Tlluc2 cells expressing non-mutated IN, while DNA-immunization with IN_in_r2 protected only 20% of mice, although tumor cells expressed IN matching the immunogen. Tumor size inversely correlated with IN-specific IFN-γ/IL-2 T-cell response. IN-expressing tumors displayed compromised metastatic activity restricted to lungs with reduced metastases size. Protective potential of IN immunogens relied on their immunogenicity for CD8+ T-cells, dependent on proteasomal processing and low level of oxidative stress.

Correction: DNA immunization site determines the level of gene expression and the magnitude, but not the type of the induced immune response.

[This corrects the article DOI: 10.1371/journal.pone.0197902.].

Frequent and Durable Anti-HIV Envelope VIV2 IgG Responses Induced by HIV-1 DNA Priming and HIV-MVA Boosting in Healthy Tanzanian Volunteers.

We evaluated antibody responses to the human immunodeficiency virus (HIV) envelope variable regions 1 and 2 (V1V2) in 29 vaccinees who had received three HIV-1 DNA immunizations and two HIV-modified vaccinia virus Ankara (MVA) boosts in the phase I/II HIVIS03 vaccine trial. Twenty vaccinees received a third HIV-MVA boost after three years in the HIVIS06 trial. IgG and IgG antibody subclasses to gp70V1V2 proteins of HIV-1 A244, CN54, Consensus C, and Case A2 were analysed using an enzyme-linked immunosorbent assay (ELISA). Cyclic V2 peptides of A244, Consensus C, and MN were used in a surface plasmon resonance (SPR) assay. Four weeks after the second HIV-MVA, anti-V1V2 IgG antibodies to A244 were detected in 97% of HIVIS03 vaccinees, in 75% three years later, and in 95% after the third HIV-MVA. Anti-CN54 V1V2 IgG was detectable in 48% four weeks after the second HIV-MVA. The SPR data supported the findings. The IgG response was predominantly IgG1. Four weeks after the second HIV-MVA, 85% of vaccinees had IgG1 antibodies to V1V2 A244, which persisted in 25% for three-years. IgG3 and IgG4 antibodies to V1V2 A244 were rare. In conclusion, the HIV-DNA/MVA vaccine regimen induced durable V1V2 IgG antibody responses in a high proportion of vaccinees.

Frequent Anti-V1V2 Responses Induced by HIV-DNA Followed by HIV-MVA with or without CN54rgp140/GLA-AF in Healthy African Volunteers.

Antibody responses that correlated with reduced risk of HIV acquisition in the RV144 efficacy trial were assessed in healthy African volunteers who had been primed three times with HIV-DNA (subtype A, B, C) and then randomized into two groups; group 1 was boosted twice with HIV-MVA (CRF01_AE) and group 2 with the same HIV-MVA coadministered with subtype C envelope (Env) protein (CN54rgp140/GLA-AF). The fine specificity of plasma Env-specific antibody responses was mapped after the final vaccination using linear peptide microarray technology. Binding IgG antibodies to the V1V2 loop in CRF01_AE and subtype C Env and Env-specific IgA antibodies were determined using enzyme-linked immunosorbent assay. Functional antibody-dependent cellular cytotoxicity (ADCC)-mediating antibody responses were measured using luciferase assay. Mapping of linear epitopes within HIV-1 Env demonstrated strong targeting of the V1V2, V3, and the immunodominant region in gp41 in both groups, with additional recognition of two epitopes located in the C2 and C4 regions in group 2. A high frequency of V1V2-specific binding IgG antibody responses was detected to CRF01_AE (77%) and subtype C antigens (65%). In conclusion, coadministration of CN54rgp140/GLA-AF with HIV-MVA did not increase the frequency, breadth, or magnitude of anti-V1V2 responses or ADCC-mediating antibodies induced by boosting with HIV-MVA alone.

Induction of Identical IgG HIV-1 Envelope Epitope Recognition Patterns After Initial HIVIS-DNA/MVA-CMDR Immunization and a Late MVA-CMDR Boost.

In the RV144 trial, to date the only HIV-1 vaccine efficacy trial demonstrating a modestly reduced risk of HIV-1 acquisition, antibody responses toward the HIV Envelope protein (Env) variable (V) 2 and V3 regions were shown to be correlated with a reduced risk of infection. These potentially protective antibody responses, in parallel with the vaccine efficacy, however, waned quickly. Dissecting vaccine-induced IgG recognition of antigenic regions and their variants within the HIV-1 Env from different vaccine trials will aid in designing future HIV-1 immunogens and vaccination schedules. We, therefore, analyzed the IgG response toward linear HIV-1 Env epitopes elicited by a multi-clade, multigene HIVIS-DNA priming, and heterologous recombinant modified vaccinia virus Ankara (MVA-CMDR) boosting regimen (HIVIS03) and assessed whether a late MVA-CMDR boost 3 years after completion of the initial vaccination schedule (HIVIS06) restored antibody responses toward these epitopes. Here we report that vaccination schedule in the HIVIS03 trial elicited IgG responses against linear epitopes within the V2 and V3 tip as well as against the gp41 immunodominant region in a high proportion of vaccinees. Antibodies against the V2 and gp41 Env regions were restricted to variants with close homology to the MVA-CMDR immunogen sequence, while V3 responses were more cross-reactive. Boosting with a late third MVA-CMDR after 3 years effectively restored waned IgG responses to linear Env epitopes and induced targeting of identical antigenic regions and variants comparable to the previous combined HIVIS-DNA/MVA-CMDR regimen. Our findings support the notion that anti-HIV-1 Env responses, associated with a reduced risk of infection in RV144, could be maintained by regular boosting with a single dose of MVA-CMDR.

Optimizing the immunogenicity of HIV prime-boost DNA-MVA-rgp140/GLA vaccines in a phase II randomized factorial trial design.

BACKGROUND: We evaluated the safety and immunogenicity of (i) an intradermal HIV-DNA regimen given with/without intradermal electroporation (EP) as prime and (ii) the impact of boosting with modified vaccinia virus Ankara (HIV-MVA) administered with or without subtype C CN54rgp140 envelope protein adjuvanted with Glucopyranosyl Lipid A (GLA-AF) in volunteers from Tanzania and Mozambique. METHODS: Healthy HIV-uninfected adults (N = 191) were randomized twice; first to one of three HIV-DNA intradermal priming regimens by needle-free ZetaJet device at weeks 0, 4 and 12 (Group I: 2x0.1mL [3mg/mL], Group II: 2x0.1mL [3mg/mL] plus EP, Group III: 1x0.1mL [6mg/mL] plus EP). Second the same volunteers received 108 pfu HIV-MVA twice, alone or combined with CN54rgp140/GLA-AF, intramuscularly by syringe, 16 weeks apart. Additionally, 20 volunteers received saline placebo. RESULTS: Vaccinations and electroporation did not raise safety concerns. After the last vaccination, the overall IFN-γ ELISpot response rate to either Gag or Env was 97%. Intradermal electroporation significantly increased ELISpot response rates to HIV-DNA-specific Gag (66% group I vs. 86% group II, p = 0.026), but not to the HIV-MVA vaccine-specific Gag or Env peptide pools nor the magnitude of responses. Co-administration of rgp140/GLA-AF with HIV-MVA did not impact the frequency of binding antibody responses against subtype B gp160, C gp140 or E gp120 antigens (95%, 99%, 79%, respectively), but significantly enhanced the magnitude against subtype B gp160 (2700 versus 300, p<0.001) and subtype C gp140 (24300 versus 2700, p<0.001) Env protein. At relatively low titers, neutralizing antibody responses using the TZM-bl assay were more frequent in vaccinees given adjuvanted protein boost. CONCLUSION: Intradermal electroporation increased DNA-induced Gag response rates but did not show an impact on Env-specific responses nor on the magnitude of responses. Co-administration of HIV-MVA with rgp140/GLA-AF significantly enhanced antibody responses.

Regulatory T cell abundance and activation status before and after priming with HIVIS-DNA and boosting with MVA-HIV/rgp140/GLA-AF may impact the magnitude of the vaccine-induced immune responses.

BACKGROUND: Little is known about regulatory CD4 T cells (Tregs) in the context of HIV vaccines. Tregs can be differentiated into resting (FoxP3+CD45RA+ - rTregs), activated (FoxP3HighCD45RA- - aTregs) and memory (FoxP3LowCD45RA- - mTregs). Tregs, as CD4 T cells, are also frequent targets for HIV infection. We studied how the abundance and phenotypes of Tregs in terms of activation status and expression of HIV-1 binding molecules would have changed during vaccination in healthy volunteers participating in a phase IIa HIV vaccine clinical trial. Subjects were primed three times with HIVIS-DNA and boosted twice with MVA-CMDR-HIV alone (n = 12) or MVA-CMDR combined with protein CN54rgp140 (n = 13). The proportions of ß7 integrin in all CD4 T cells and in the Tregs subset decreased moderately after the final vaccination (p = 0.001 and p = 0.033, respectively) and the rTregs proportion within the total Tregs were also decreased after the final vaccination (p = 0.038). All these proportions returned to normal values within the three months after the final vaccination. The magnitude of HIV-Envelope-specific IFNγ + T cells after vaccination (r = 0.66; p = 0.021) correlated directly with the proportion of Tregs, and correlated inversely correlated with ratios of Th17/Tregs (r = -0.75; p = 0.0057) and Th17/mTregs (r = -0.78; p = 0.0065). Higher titers of IgG gp140 antibodies were observed in subjects with higher mTregs proportions (r = 0.52; p = 0.022). Interestingly, pre-vaccination levels of mTregs correlated with vaccine-induced Env-binding antibodies (r = 0.57; p = 0.01) and presence of neutralizing antibodies (r = 0.61; p = 0.01), while the pre-vaccination Th17/mTregs ratio correlated inversely with the magnitude of cellular IFN-γ ELISpot responses (r = -0.9; p = 0.002). Taken together, these results suggest that pre- and post-vaccination Tregs, their activation status, the Th17/Tregs ratio and other host factors affecting Treg abundance, have an impact on the magnitude of HIV vaccine-induced immune responses. Moreover, the DNA-HIVIS/MVA-HIV regimen, alone or in combination with CN54rgp140 induced moderate and temporary alterations of the Tregs activation status. We also show a decrease in expression of the HIV-1 ligand ß7 integrin on Tregs and all CD4 T cells.

DNA immunization site determines the level of gene expression and the magnitude, but not the type of the induced immune response.

Optimization of DNA vaccine delivery improves the potency of the immune response and is crucial to clinical success. Here, we inquired how such optimization impacts the magnitude of the response, its specificity and type. BALB/c mice were DNA-immunized with two model immunogens, HIV-1 protease and reverse transcriptase by intramuscular or intradermal injections with electroporation. DNA immunogens were co-delivered with DNA encoding luciferase. Delivery and expression were monitored by in vivo bioluminescence imaging (BLI). The endpoint immune responses were assessed by IFN-γ/IL-2 FluoroSpot, multiparametric flow cytometry and antibody ELISA. Expression and immunogenicity were compared in relation to the delivery route. Regardless of the route, protease generated mainly IFN-γ, and reverse transcriptase, IL-2 and antibody response. BLI of mice immunized with protease- or reverse transcriptase/reporter plasmid mixtures, demonstrated significant loss of luminescence over time. The rate of decline of luminescence strongly correlated with the magnitude of immunogen-specific response, and depended on the immunogenicity profile and the immunization route. In vitro and in vivo BLI-based assays demonstrated that intradermal delivery strongly improved the immunogenicity of protease, and to a lesser extent, of reverse transcriptase. Immune response polarization and epitope hierarchy were not affected. Thus, by changing delivery/immunogen expression sites, it is possible to modulate the magnitude, but not the type or fine specificity of the induced immune response.

Intradermal HIV-1 DNA Immunization Using Needle-Free Zetajet Injection Followed by HIV-Modified Vaccinia Virus Ankara Vaccination Is Safe and Immunogenic in Mozambican Young Adults: A Phase I Randomized Controlled Trial.

We assessed the safety and immunogenicity of HIV-DNA priming using Zetajet™, a needle-free device intradermally followed by intramuscular HIV-MVA boosts, in 24 healthy Mozambicans. Volunteers were randomized to receive three immunizations of 600 µg (n = 10; 2 × 0.1 ml) or 1,200 µg (n = 10; 2 × 0.2 ml) of HIV-DNA (3 mg/ml), followed by two boosts of 108 pfu HIV-MVA. Four subjects received placebo saline injections. Vaccines and injections were safe and well tolerated with no difference between the two priming groups. After three HIV-DNA immunizations, IFN-γ ELISpot responses to Gag were detected in 9/17 (53%) vaccinees, while none responded to Envelope (Env). After the first HIV-MVA, the overall response rate to Gag and/or Env increased to 14/15 (93%); 14/15 (93%) to Gag and 13/15 (87%) to Env. There were no significant differences between the immunization groups in frequency of response to Gag and Env or magnitude of Gag responses. Env responses were significantly higher in the higher dose group (median 420 vs. 157.5 SFC/million peripheral blood mononuclear cell, p = .014). HIV-specific antibodies to subtype C gp140 and subtype B gp160 were elicited in all vaccinees after the second HIV-MVA, without differences in titers between the groups. Neutralizing antibody responses were not detected. Two (13%) of 16 vaccinees, one in each of the priming groups, exhibited antibodies mediating antibody-dependent cellular cytotoxicity to CRF01_AE. In conclusion, HIV-DNA vaccine delivered intradermally in volumes of 0.1-0.2 ml using Zetajet was safe and well tolerated. Priming with the 1,200 µg dose of HIV-DNA generated higher magnitudes of ELISpot responses to Env.

Preferential Targeting of Conserved Gag Regions after Vaccination with a Heterologous DNA Prime-Modified Vaccinia Virus Ankara Boost HIV-1 Vaccine Regimen.

Prime-boost vaccination strategies against HIV-1 often include multiple variants for a given immunogen for better coverage of the extensive viral diversity. To study the immunologic effects of this approach, we characterized breadth, phenotype, function, and specificity of Gag-specific T cells induced by a DNA-prime modified vaccinia virus Ankara (MVA)-boost vaccination strategy, which uses mismatched Gag immunogens in the TamoVac 01 phase IIa trial. Healthy Tanzanian volunteers received three injections of the DNA-SMI vaccine encoding a subtype B and AB-recombinant Gagp37 and two vaccinations with MVA-CMDR encoding subtype A Gagp55 Gag-specific T-cell responses were studied in 42 vaccinees using fresh peripheral blood mononuclear cells. After the first MVA-CMDR boost, vaccine-induced gamma interferon-positive (IFN-γ+) Gag-specific T-cell responses were dominated by CD4+ T cells (P < 0.001 compared to CD8+ T cells) that coexpressed interleukin-2 (IL-2) (66.4%) and/or tumor necrosis factor alpha (TNF-α) (63.7%). A median of 3 antigenic regions were targeted with a higher-magnitude median response to Gagp24 regions, more conserved between prime and boost, compared to those of regions within Gagp15 (not primed) and Gagp17 (less conserved; P < 0.0001 for both). Four regions within Gagp24 each were targeted by 45% to 74% of vaccinees upon restimulation with DNA-SMI-Gag matched peptides. The response rate to individual antigenic regions correlated with the sequence homology between the MVA- and DNA Gag-encoded immunogens (P = 0.04, r2 = 0.47). In summary, after the first MVA-CMDR boost, the sequence-mismatched DNA-prime MVA-boost vaccine strategy induced a Gag-specific T-cell response that was dominated by polyfunctional CD4+ T cells and that targeted multiple antigenic regions within the conserved Gagp24 protein.IMPORTANCE Genetic diversity is a major challenge for the design of vaccines against variable viruses. While including multiple variants for a given immunogen in prime-boost vaccination strategies is one approach that aims to improve coverage for global virus variants, the immunologic consequences of this strategy have been poorly defined so far. It is unclear whether inclusion of multiple variants in prime-boost vaccination strategies improves recognition of variant viruses by T cells and by which mechanisms this would be achieved, either by improved cross-recognition of multiple variants for a given antigenic region or through preferential targeting of antigenic regions more conserved between prime and boost. Engineering vaccines to induce adaptive immune responses that preferentially target conserved antigenic regions of viral vulnerability might facilitate better immune control after preventive and therapeutic vaccination for HIV and for other variable viruses.

Three-Year Durability of Immune Responses Induced by HIV-DNA and HIV-Modified Vaccinia Virus Ankara and Effect of a Late HIV-Modified Vaccinia Virus Ankara Boost in Tanzanian Volunteers.

We explored the duration of immune responses and the effect of a late third HIV-modified vaccinia virus Ankara (MVA) boost in HIV-DNA primed and HIV-MVA boosted Tanzanian volunteers. Twenty volunteers who had previously received three HIV-DNA and two HIV-MVA immunizations were given a third HIV-MVA immunization 3 years after the second HIV-MVA boost. At the time of the third HIV-MVA, 90% of the vaccinees had antibodies to HIV-1 subtype C gp140 (median titer 200) and 85% to subtype B gp160 (median titer 100). The majority of vaccinees had detectable antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies, 70% against CRF01_AE virus-infected cells (median titer 239) and 84% against CRF01_AE gp120-coated cells (median titer 499). A high proportion (74%) of vaccinees had IFN-γ ELISpot responses, 63% to Gag and 42% to Env, 3 years after the second HIV-MVA boost. After the third HIV-MVA, there was an increase in Env-binding antibodies and ADCC-mediating antibodies relative to the response seen at the time of the third HIV-MVA vaccination, p < .0001 and p < .05, respectively. The frequency of IFN-γ ELISpot responses increased to 95% against Gag or Env and 90% to both Gag and Env, p = .064 and p = .002, respectively. In conclusion, the HIV-DNA prime/HIV-MVA boost regimen elicited potent antibody and cellular immune responses with remarkable durability, and a third HIV-MVA immunization significantly boosted both antibody and cellular immune responses relative to the levels detected at the time of the third HIV-MVA, but not to higher levels than after the second HIV-MVA.

Boosting with Subtype C CN54rgp140 Protein Adjuvanted with Glucopyranosyl Lipid Adjuvant after Priming with HIV-DNA and HIV-MVA Is Safe and Enhances Immune Responses: A Phase I Trial.

BACKGROUND: A vaccine against HIV is widely considered the most effective and sustainable way of reducing new infections. We evaluated the safety and impact of boosting with subtype C CN54rgp140 envelope protein adjuvanted in glucopyranosyl lipid adjuvant (GLA-AF) in Tanzanian volunteers previously given three immunizations with HIV-DNA followed by two immunizations with recombinant modified vaccinia virus Ankara (HIV-MVA). METHODS: Forty volunteers (35 vaccinees and five placebo recipients) were given two CN54rgp140/GLA-AF immunizations 30-71 weeks after the last HIV-MVA vaccination. These immunizations were delivered intramuscularly four weeks apart. RESULTS: The vaccine was safe and well tolerated except for one episode of asymptomatic hypoglycaemia that was classified as severe adverse event. Two weeks after the second HIV-MVA vaccination 34 (97%) of the 35 previously vaccinated developed Env-specific binding antibodies, and 79% and 84% displayed IFN-γ ELISpot responses to Gag and Env, respectively. Binding antibodies to subtype C Env (included in HIV-DNA and protein boost), subtype B Env (included only in HIV-DNA) and CRF01_AE Env (included only in HIV-MVA) were significantly boosted by the CN54rgp140/GLA-AF immunizations. Functional antibodies detected using an infectious molecular clone virus/peripheral blood mononuclear cell neutralization assay, a pseudovirus/TZM-bl neutralization assay or by assays for antibody-dependent cellular cytotoxicity (ADCC) were not significantly boosted. In contrast, T-cell proliferative responses to subtype B MN antigen and IFN-γ ELISpot responses to Env peptides were significantly enhanced. Four volunteers not primed with HIV-DNA and HIV-MVA before the CN54rgp140/GLA-AF immunizations mounted an antibody response, while cell-mediated responses were rare. After the two Env subtype C protein immunizations, a trend towards higher median subtype C Env binding antibody titers was found in vaccinees who had received HIV-DNA and HIV-MVA prior to the two Env protein immunizations as compared to unprimed vaccinees (p = 0.07). CONCLUSION: We report excellent tolerability, enhanced binding antibody responses and Env-specific cell-mediated immune responses but no ADCC antibody increase after two immunizations with a subtype C rgp140 protein adjuvanted in GLA-AF in healthy volunteers previously immunized with HIV-DNA and HIV-MVA. TRIAL REGISTRATION: International Clinical Trials Registry PACTR2010050002122368.

Early antiretroviral therapy in children perinatally infected with HIV: a unique opportunity to implement immunotherapeutic approaches to prolong viral remission.

From the use of antiretroviral therapy to prevent mother-to-child transmission to the possibility of HIV cure hinted at by the Mississippi baby experience, paediatric HIV infection has been pivotal to our understanding of HIV pathogenesis and management. Daily medication and indefinite antiretroviral therapy is recommended for children infected with HIV. Maintenance of life-long adherence is difficult and the incidence of triple-class virological failure after initiation of antiretroviral therapy increases with time. This challenge shows the urgent need to define novel strategies to provide long-term viral suppression that will allow safe interruption of antiretroviral therapy without viral rebound and any associated complications. HIV-infected babies treated within a few days of birth have a unique combination of a very small pool of integrated viruses, a very high proportion of relatively HIV resistant naive T cells, and an unparalleled capacity to regenerate an immune repertoire. These features make this group the optimum model population to investigate the potential efficacy of immune-based therapies. If successful, these investigations could change the way we manage HIV infection.

HIV-DNA Given with or without Intradermal Electroporation Is Safe and Highly Immunogenic in Healthy Swedish HIV-1 DNA/MVA Vaccinees: A Phase I Randomized Trial.

BACKGROUND: We compared safety and immunogenicity of intradermal (ID) vaccination with and without electroporation (EP) in a phase I randomized placebo-controlled trial of an HIV-DNA prime HIV-MVA boost vaccine in healthy Swedish volunteers. METHODS: HIV-DNA plasmids encoding HIV-1 genes gp160 subtypes A, B and C; Rev B; Gag A and B and RTmut B were given ID at weeks 0, 6 and 12 in a dose of 0.6 mg. Twenty-five volunteers received vaccine using a needle-free device (ZetaJet) with (n=16) or without (n=9) ID EP (Dermavax). Five volunteers were placebo recipients. Boosting with recombinant MVA-CMDR expressing HIV-1 Env, Gag, Pol of CRF01_AE (HIV-MVA) or placebo was performed at weeks 24 and 40. Nine of the vaccinees received a subtype C CN54 gp140 protein boost together with HIV-MVA. RESULTS: The ID/EP delivery was very well tolerated. After three HIV-DNA immunizations, no statistically significant difference was seen in the IFN-γ ELISpot response rate to Gag between HIV-DNA ID/EP recipients (5/15, 33%) and HIV-DNA ID recipients (1/7, 14%, p=0.6158). The first HIV-MVA or HIV-MVA+gp140 vaccination increased the IFN-γ ELISpot response rate to 18/19 (95%). CD4+ and/or CD8+ T cell responses to Gag or Env were demonstrable in 94% of vaccinees. A balanced CD4+ and CD8+ T cell response was noted, with 78% and 71% responders, respectively. IFN-γ and IL-2 dominated the CD4+ T cell response to Gag and Env. The CD8+ response to Gag was broader with expression of IFN-γ, IL-2, MIP-1ß and/or CD107. No differences were seen between DNA vaccine groups. Binding antibodies were induced after the second HIV-MVA+/-gp140 in 93% of vaccinees to subtype C Env, with the highest titers among EP/gp140 recipients. CONCLUSION: Intradermal electroporation of HIV-DNA was well tolerated. Strong cell- and antibody-mediated immune responses were elicited by the HIV-DNA prime and HIV-MVA boosting regimen, with or without intradermal electroporation use. TRIAL REGISTRATION: International Standard Randomised Controlled Trial Number (ISRCTN) 60284968.

Priming with a simplified intradermal HIV-1 DNA vaccine regimen followed by boosting with recombinant HIV-1 MVA vaccine is safe and immunogenic: a phase IIa randomized clinical trial.

BACKGROUND: Intradermal priming with HIV-1 DNA plasmids followed by HIV-1MVA boosting induces strong and broad cellular and humoral immune responses. In our previous HIVIS-03 trial, we used 5 injections with 2 pools of HIV-DNA at separate sites for each priming immunization. The present study explores whether HIV-DNA priming can be simplified by reducing the number of DNA injections and administration of combined versus separated plasmid pools. METHODS: In this phase IIa, randomized trial, priming was performed using 5 injections of HIV-DNA, 1000 µg total dose, (3 Env and 2 Gag encoding plasmids) compared to two "simplified" regimens of 2 injections of HIV-DNA, 600 µg total dose, of Env- and Gag-encoding plasmid pools with each pool either administered separately or combined. HIV-DNA immunizations were given intradermally at weeks 0, 4, and 12. Boosting was performed intramuscularly with 108 pfu HIV-MVA at weeks 30 and 46. RESULTS: 129 healthy Tanzanian participants were enrolled. There were no differences in adverse events between the groups. The proportion of IFN-γ ELISpot responders to Gag and/or Env peptides after the second HIV-MVA boost did not differ significantly between the groups primed with 2 injections of combined HIV-DNA pools, 2 injections with separated pools, and 5 injections with separated pools (90%, 97% and 97%). There were no significant differences in the magnitude of Gag and/or Env IFN-γ ELISpot responses, in CD4+ and CD8+ T cell responses measured as IFN-γ/IL-2 production by intracellular cytokine staining (ICS) or in response rates and median titers for binding antibodies to Env gp160 between study groups. CONCLUSIONS: A simplified intradermal vaccination regimen with 2 injections of a total of 600 µg with combined HIV-DNA plasmids primed cellular responses as efficiently as the standard regimen of 5 injections of a total of 1000 µg with separated plasmid pools after boosting twice with HIV-MVA. TRIAL REGISTRATION: World Health Organization International Clinical Trials Registry Platform PACTR2010050002122368.

Potent functional antibody responses elicited by HIV-I DNA priming and boosting with heterologous HIV-1 recombinant MVA in healthy Tanzanian adults.

UNLABELLED: Vaccine-induced HIV antibodies were evaluated in serum samples collected from healthy Tanzanian volunteers participating in a phase I/II placebo-controlled double blind trial using multi-clade, multigene HIV-DNA priming and recombinant modified vaccinia Ankara (HIV-MVA) virus boosting (HIVIS03). The HIV-DNA vaccine contained plasmids expressing HIV-1 gp160 subtypes A, B, C, Rev B, Gag A, B and RTmut B, and the recombinant HIV-MVA boost expressed CRF01_AE HIV-1 Env subtype E and Gag-Pol subtype A. While no neutralizing antibodies were detected using pseudoviruses in the TZM-bl cell assay, this prime-boost vaccination induced neutralizing antibodies in 83% of HIVIS03 vaccinees when a peripheral blood mononuclear cell (PBMC) assay using luciferase reporter-infectious molecular clones (LucR-IMC) was employed. The serum neutralizing activity was significantly (but not completely) reduced upon depletion of natural killer (NK) cells from PBMC (p=0.006), indicating a role for antibody-mediated Fcγ-receptor function. High levels of antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies against CRF01_AE and/or subtype B were subsequently demonstrated in 97% of the sera of vaccinees. The magnitude of ADCC-mediating antibodies against CM235 CRF01_AE IMC-infected cells correlated with neutralizing antibodies against CM235 in the IMC/PBMC assay. In conclusion, HIV-DNA priming, followed by two HIV-MVA boosts elicited potent ADCC responses in a high proportion of Tanzanian vaccinees. Our findings highlight the potential of HIV-DNA prime HIV-MVA boost vaccines for induction of functional antibody responses and suggest this vaccine regimen and ADCC studies as potentially important new avenues in HIV vaccine development. TRIAL REGISTRATION: Controlled-Trials ISRCTN90053831 The Pan African Clinical Trials Registry ATMR2009040001075080 (currently PACTR2009040001075080).

Short communication: HIV-1 Nef protein carries multiple epitopes suitable for induction of cellular immunity for an HIV vaccine in Africa.

Using the early protein HIV Nef, new HLA class I binding epitopes of importance for immune responses to HIV were predicted for common African alleles. In total we identified 45 epitopes previously not described for the HLA alleles A*30:01, A*30:02, B*58:01, and C*07:01 and compared them to reported epitopes, primarily from HLA-A*02:01, from the Los Alamos database and our own vaccine studies. Related to its small size, the Nef gene/protein appears to be able to contribute effectively to confer both stronger and broader cellular immunogenicity to an HIV-1 vaccine. We also propose feasible mutations of such an additional vaccine antigen to preserve its immunogenicity, modified not to confer HLA or CD4(+) down-regulating activities. This article includes data on a valuable HIV immunogenic component for a vaccine in Africa.