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.

Broad and potent cellular and humoral immune responses after a second late HIV-modified vaccinia virus ankara vaccination in HIV-DNA-primed and HIV-modified vaccinia virus Ankara-boosted Swedish vaccinees.

We have previously shown that an HIV vaccine regimen including three HIV-DNA immunizations and a single HIV-modified vaccinia virus Ankara (MVA) boost was safe and highly immunogenic in Swedish volunteers. A median 38 months after the first HIV-MVA vaccination, 24 volunteers received 10(8) plaque-forming units of HIV-MVA. The vaccine was well tolerated. Two weeks after this HIV-MVA vaccination, 18 (82%) of 22 evaluable vaccinees were interferon (IFN)-γ enzyme-linked immunospot (ELISpot) reactive: 18 to Gag and 10 (45%) to Env. A median minimal epitope count of 4 to Gag or Env was found in a subset of 10 vaccinees. Intracellular cytokine staining revealed CD4(+) and/or CD8(+) T cell responses in 23 (95%) of 24 vaccinees, 19 to Gag and 19 to Env. The frequency of HIV-specific CD4(+) and CD8(+) T cell responses was equally high (75%). A high proportion of CD4(+) and CD8(+) T cell responses to Gag was polyfunctional with production of three or more cytokines (40% and 60%, respectively). Of the Env-specific CD4(+) T cells 40% were polyfunctional. Strong lymphoproliferative responses to Aldrithiol-2 (AT-2)-treated subtype A, B, C, and A_E virus were demonstrable in 21 (95%) of 22 vaccinees. All vaccinees developed binding antibodies to Env and Gag. Neutralizing antibodies were detected in a peripheral blood mononuclear cell (PBMC)-based assay against subtype B and CRF01_AE viruses. The neutralizing antibody response rates were influenced by the vaccine dose and/or mode of delivery used at the previous HIV-MVA vaccination. Thus, a second late HIV-MVA boost induced strong and broad cellular immune responses and improved antibody responses. The data support further exploration of this vaccine concept.

Broad and potent immune responses to a low dose intradermal HIV-1 DNA boosted with HIV-1 recombinant MVA among healthy adults in Tanzania.

BACKGROUND: We conducted a phase I/II randomized placebo-controlled trial with the aim of exploring whether priming with a low intradermal dose of a multiclade, multigene HIV-1 DNA vaccine could improve the immunogenicity of the same vaccine given intramuscularly prior to boosting with a heterologous HIV-1 MVA among healthy adults in Dar es Salaam, Tanzania. METHODS: Sixty HIV-uninfected volunteers were randomized to receive DNA plasmid vaccine 1mg intradermally (id), n=20, or 3.8mg intramuscularly (im), n=20, or placebo, n=20, using a needle-free injection device. DNA plasmids encoding HIV-1 genes gp160 subtype A, B, C; rev B; p17/p24 gag A, B and Rtmut B were given at weeks 0, 4 and 12. Recombinant MVA (10(8)pfu) expressing HIV-1 Env, Gag, Pol of CRF01_AE or placebo was administered im at month 9 and 21. RESULTS: The vaccines were well tolerated. Two weeks after the third HIV-DNA injection, 22/38 (58%) vaccinees had IFN-γ ELISpot responses to Gag. Two weeks after the first HIV-MVA boost all 35 (100%) vaccinees responded to Gag and 31 (89%) to Env. Two to four weeks after the second HIV-MVA boost, 28/29 (97%) vaccinees had IFN-γ ELISpot responses, 27 (93%) to Gag and 23 (79%) to Env. The id-primed recipients had significantly higher responses to Env than im recipients. Intracellular cytokine staining for Gag-specific IFN-γ/IL-2 production showed both CD8(+) and CD4(+) T cell responses. All vaccinees had HIV-specific lymphoproliferative responses. All vaccinees reacted in diagnostic HIV serological tests and 26/29 (90%) had antibodies against gp160 after the second HIV-MVA boost. Furthermore, while all of 29 vaccinee sera were negative for neutralizing antibodies against clade B, C and CRF01_AE pseudoviruses in the TZM-bl neutralization assay, in a PBMC assay, the response rate ranged from 31% to 83% positives, depending upon the clade B or CRF01_AE virus tested. CONCLUSIONS: This vaccine approach is safe and highly immunogenic. Low dose, id HIV-DNA priming elicited higher and broader cell-mediated immune responses to Env after HIV-MVA boost compared to a higher HIV-DNA priming dose given im. Three HIV-DNA priming immunizations followed by two HIV-MVA boosts efficiently induced Env-antibody responses.

Treatment of Kaposi sarcoma in human immunodeficiency virus-1-infected Mozambican children with antiretroviral drugs and chemotherapy.

AIDS-associated Kaposi sarcoma occurs in children, but treatment experience reports are very scarce. A retrospective analysis of 28 children treated with highly active antiretroviral therapy and monthly paclitaxel showed unexpected results with 19 children in complete and sustainable remission, including those with the most severe form. Tolerance and feasibility were good, despite a lack of skilled staff in a low-resource setting.

Immunization with recombinant HLA classes I and II, HIV-1 gp140, and SIV p27 elicits protection against heterologous SHIV infection in rhesus macaques.

Major histocompatibility complex (MHC) molecules expressed on the surface of human immunodeficiency virus (HIV) are potential targets for neutralizing antibodies. Since MHC molecules are polymorphic, nonself MHC can also be immunogenic. We have used combinations of novel recombinant HLA class I and II and HIV/simian immunodeficiency virus (SIV) antigens, all linked to dextran, to investigate whether they can elicit protective immunity against heterologous simian/human immunodeficiency virus (SHIV) challenge in rhesus macaques. Three groups of animals were immunized with HLA (group 1, n = 8), trimeric YU2 HIV type 1 (HIV-1) gp140 and SIV p27 (HIV/SIV antigens; group 2, n = 8), or HLA plus HIV/SIV antigens (group 3, n = 8), all with Hsp70 and TiterMax Gold adjuvant. Another group (group 4, n = 6) received the same vaccine as group 3 without TiterMax Gold. Two of eight macaques in group 3 were completely protected against intravenous challenge with 18 50% animal infective doses (AID(50)) of SHIV-SF162P4/C grown in human cells expressing HLA class I and II lineages represented in the vaccine, while the remaining six macaques showed decreased viral loads compared to those in unimmunized animals. Complement-dependent neutralizing activity in serum and high levels of anti-HLA antibodies were elicited in groups 1 and 3, and both were inversely correlated with the plasma viral load at 2 weeks postchallenge. Antibody-mediated protection was strongly supported by the fact that transfer of pooled serum from the two challenged but uninfected animals protected two naïve animals against repeated low-dose challenge with the same SHIV stock. This study demonstrates that immunization with recombinant HLA in combination with HIV-1 antigens might be developed into an alternative strategy for a future AIDS vaccine.

Biodistribution, persistence and lack of integration of a multigene HIV vaccine delivered by needle-free intradermal injection and electroporation.

It is likely that gene-based vaccines will enter the human vaccine area soon. A few veterinary vaccines employing this concept have already been licensed, and a multitude of clinical trials against infectious diseases or different forms of cancer are ongoing. Highly important when developing novel vaccines are the safety aspects and also new adjuvants and delivery techniques needs to be carefully investigated so that they meet all short- and long-term safety requirements. One novel in vivo delivery method for plasmid vaccines is electroporation, which is the application of short pulses of electric current immediately after, and at the site of, an injection of a genetic vaccine. This method has been shown to significantly augment the transfection efficacy and the subsequent vaccine-specific immune responses. However, the dramatic increase in delivery efficacy offered by electroporation has raised concerns of potential increase in the risk of integration of plasmid DNA into the host genome. Here, we demonstrate the safety and lack of integration after immunization with a high dose of a multigene HIV-1 vaccine delivered intradermally using the needle free device Biojector 2000 together with electroporation using Derma Vax™ DNA Vaccine Skin Delivery System. We demonstrate that plasmids persist in the skin at the site of injection for at least four months after immunization. However, no association between plasmid DNA and genomic DNA could be detected as analyzed by qPCR following field inversion gel electrophoresis separating heavy and light DNA fractions. We will shortly initiate a phase I clinical trial in which healthy volunteers will be immunized with this multiplasmid HIV-1 vaccine using a combination of the delivery methods jet-injection and intradermal electroporation.

Strong HIV-specific CD4+ and CD8+ T-lymphocyte proliferative responses in healthy individuals immunized with an HIV-1 DNA vaccine and boosted with recombinant modified vaccinia virus ankara expressing HIV-1 genes.

We investigated HIV-1 vaccine-induced lymphoproliferative responses in healthy volunteers immunized intradermally or intramuscularly (with or without adjuvant granulocyte-macrophage colony-stimulating factor [GM-CSF] protein) with DNA expressing HIV-1 gag, env, rev, and rt at months 0, 1, and 3 using a Biojector and boosted at 9 months with modified vaccinia virus Ankara (MVA) expressing heterologous HIV-1 gag, env, and pol (HIV-MVA). Lymphoproliferative responses to aldrithiol-2 (AT-2)-inactivated-HIV-1 antigen were tested by a [(3)H]thymidine uptake assay and a flow-cytometric assay of specific cell-mediated immune response in activated whole blood (FASCIA-WB) 2 weeks after the HIV-MVA boost (n = 38). A FASCIA using peripheral blood mononuclear cells (FASCIA-PBMC) was also employed (n = 14). Thirty-five of 38 (92%) vaccinees were reactive by the [(3)H]thymidine uptake assay. Thirty-two of 38 (84%) vaccinees were reactive by the CD4(+) T-cell FASCIA-WB, and 7 of 38 (18%) also exhibited CD8(+) T-cell responses. There was strong correlation between the proliferative responses measured by the [(3)H]thymidine uptake assay and CD4(+) T-cell FASCIA-WB (r = 0.68; P < 0.01). Fourteen vaccinees were analyzed using all three assays. Ten of 14 (71%) and 11/14 (79%) demonstrated CD4(+) T-cell responses in FASCIA-WB and FASCIA-PBMC, respectively. CD8(+) T-cell reactivity was observed in 3/14 (21%) and 7/14 (50%) using the FASCIA-WB and FASCIA-PBMC, respectively. All 14 were reactive by the [(3)H]thymidine uptake assay. The overall HIV-specific T-cell proliferative response in the vaccinees employing any of the assays was 100% (38/38). A standardized FASCIA-PBMC, which allows simultaneous phenotyping, may be an option to the [(3)H]thymidine uptake assay for assessment of vaccine-induced T-cell proliferation, especially in isotope-restricted settings.

Nonvertical, nonsexual transmission of human immunodeficiency virus in children.

In a cohort of 450 human immunodeficiency virus (HIV)-infected children followed up in Maputo, Mozambique, 22 were born to HIV-uninfected mothers and had no history of sexual abuse. A case record review of these nonvertically, nonsexually infected children as well as a case-control study strongly suggests health care as possible source of infection for many of these children. This facet of the pediatric HIV epidemic should not be overlooked.

Broad immunogenicity of a multigene, multiclade HIV-1 DNA vaccine boosted with heterologous HIV-1 recombinant modified vaccinia virus Ankara.

BACKGROUND: A human immunodeficiency virus (HIV) vaccine that limits disease and transmission is urgently needed. This clinical trial evaluated the safety and immunogenicity of an HIV vaccine that combines a plasmid-DNA priming vaccine and a modified vaccinia virus Ankara (MVA) boosting vaccine. METHODS: Forty healthy volunteers were injected with DNA plasmids containing gp160 of HIV-1 subtypes A, B, and C; rev B; p17/p24 gag A and B, and RTmut B by use of a needle-free injection system. The vaccine was administered intradermally or intramuscularly, with or without recombinant granulocyte macrophage colony-stimulating factor, and boosted with a heterologous MVA containing env, gag, and pol of CRF01A_E. Immune responses were monitored with HIV-specific interferon (IFN)-gamma and interleukin (IL)-2 ELISpot and lymphoproliferative assays (LPAs). RESULTS: Vaccine-related adverse events were mild and tolerable. After receipt of the DNA priming vaccine, 11 (30%) of 37 vaccinees had HIV-specific IFN-gamma responses. After receipt of the MVA boosting vaccine, ELISpot assays showed that 34 (92%) of 37 vaccinees had HIV-specific IFN-gamma responses, 32 (86%) to Gag and 24 (65%) to Env. IFN-gamma production was detected in both the CD8(+) T cell compartment (5 of 9 selected vaccinees) and the CD4(+) T cell compartment (9 of 9). ELISpot results showed that 25 (68%) of 37 vaccinees had a positive IL-2 response and 35 (92%) of 38 had a positive LPA response. Of 38 subjects, a total of 37 (97%) were responders. One milligram of HIV-1 DNA administered intradermally was as effective as 4 mg administered intramuscularly in priming for the MVA boosting vaccine. CONCLUSION: This HIV-DNA priming-MVA boosting approach is safe and highly immunogenic. TRIALS REGISTRATION: International Standard Randomised Controlled Trial number: ISRCTN32604572 .

A novel assay for assessment of HIV-specific cytotoxicity by multiparameter flow cytometry.

BACKGROUND: Assessment of CD8(+) T-cell activity is of significant importance for the evaluation of cellular immune responses to viral infections, especially in HIV. We present a new assay for the assessment of HIV-specific cytotoxicity by multiparameter flow cytometry. METHODS: Target cells, pulsed with peptide pools (Gag or Nef), were stained with 5- (and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), cultured with specific or nonspecific effector cells, and finally stained with propidium iodide (PI). Determination of cytolysis is based on the enumeration of viable target cells (CFSE(hi)PI(-)) in the test sample (target and specific effector cells) as compared with that of the viable target cells in the control sample (target and nonspecific effector cells). The (51)Cr-release assay and IFN-gamma ELISpot were performed by standard procedures. RESULTS: A comparison with the Cr-release showed that the two assays were strongly correlated (r = 0.67; P < 0.001) but the sensitivity of the flow cytometric assay was significantly higher (P < 0.05), and the reproducibility good (CV, 7.7%). Good correlation was also found with the ELISpot assay (r = 0.66; P < 0.01). CONCLUSION: This new assay provides both specific and sensitive results when employed for the detection of HIV-specific CTL and can be a valuable tool for the evaluation of cytolytic activity in vaccine trials or in HIV-infected subjects, especially if such responses are present at low levels.

The prevalence and pattern of skin diseases in relation to CD4 counts among HIV-infected police officers in Dar es Salaam.

Among HIV-infected individuals, skin diseases cause significant morbidity and are frequently the initial indication of immunosuppression. From an on-going cohort study to determine prevalence and incidence of HIV infection among police officers (POs) and their suitability for HIV vaccine trials, a sub-study was carried out to determine the prevalence and pattern of skin diseases among HIV-infected POs and relate this to their immunodeficiency status. Consenting HIV-infected POs and their age and sex-matched HIV-negative officers were assessed for presence and type of skin diseases at their workplaces. A questionnaire was used for data collection. Immunodeficiency was measured by plasma CD4+ lymphocytes using flow cytometry. Between November 1998 and 31 December 2000, 716 POs were assessed. Overall HIV-1 prevalence was 17.7% (127/716). One hundred and ninety-one POs (26.7%) had at least one skin diagnosis. HIV-infected POs had significantly higher (41.7%) prevalence of skin diseases than HIV-uninfected POs (26.4%), P = 0.002. Fungal infections were common in both HIV-infected and uninfected POs. Among the HIV infected, other common diseases were: herpes zoster (11.8%); pruritic papular eruption (PPE) (7.1%); seborrheic dermatitis (5.5%); and Kaposi's sarcoma (KS) (1.6%). KS and PPE were associated with severe immunodeficiency, with mean absolute (percentage) CD4+ counts of 75.5 cells/microL (4.0%) and 71.7 cells/microL (4.8%), respectively. The values for herpes zoster and seborrheic dermatitis were 271.1 cells/micronL (12.4%) and 206.3 cells/microL (11.3%), respectively. Skin diseases were common among HIV-infected POs. PPE and KS are markers of severe immunodeficiency due to HIV. PPE, herpes zoster and KS strongly suggest underlying HIV-related immunodeficiency and patients with these conditions should be counselled and tested for HIV.