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Background National vaccine coverage in Sweden is high. Recurrent outbreaks of measles and rubella however highlight that there are immunity gaps in the population. Current knowledge about immunization status of undocumented migrant children is scant. The WHO/Europe has developed the Guide to tailoring immunization programmes (TIP) to assist countries in diagnosing barriers and motivators to vaccination in communities with low vaccination coverage. This study explored determinants to vaccination among undocumented immigrants, using the TIP guide. Methods This was qualitative study featuring interviews of seven undocumented parents recruited at nongovernmental clinics; three nurses at Child Health Centers; and information from key stakeholders retrieved at workshops as part of the TIP process. Findings The content analysis revealed two main themes: parental fear of being questioned and parental acceptance of child immunization. Undocumented parents had a positive view and attitude towards childhood immunization but expressed strong fear of being asked for identification papers at health care facilities. Frequent mobility among undocumented may limit access to complete immunization schedule. Undocumented parents mistrust health care providers and avoid health facilities, further delaying childrens' access to health care, including immunization services. Interpretention The findings of this study confirm the complexity of barriers that undocumented parents face regarding childhood immunization. The TIP guide offers a valuable process for mixed data collection and analysis, for a deeper understanding of the determinants of immunization challenges among undocumented migrants. Funding: The Public Health Agency of Sweden and WHO/Europe. Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: The study received ethical approval for the interviews from the Regional Ethics Committee in Stockholm, Sweden, Dnr 2013/678-31/3.

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.

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-1β 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

The assessment of cytokine production is an important component of studies of cell-mediated immune responses (CMI) to immunological challenges. In this study, we present a method to enhance the detection of cytokine-producing cells by allowing antigen-specific cells to expand in long-term culture. We investigated the influence of the degree of dilution of whole blood and the duration of the incubation period on whole blood as well as peripheral blood mononuclear cells (PBMCs), cultured in the absence or presence of mitogens, superantigens or specific antigens, for intracellular cytokine production (IFNgamma, TNFalpha, IL-2, IL-4, IL-10 and IL-13) by CD4+ and CD8+ T lymphocytes using four-colour flow cytometry. Whole blood was diluted 1/1, 1/2, 1/5 and 1/10, and cultured for 6, 24, 48, 72 and 120 h in the presence of antibodies against the co-stimulatory molecules CD28 and CD49d, and, during the last 4 h of culture, in the presence of brefeldin A. Optimum conditions for detection of a high number of IFNgamma-positive cells were observed after 72 h of culture in blood diluted 1/10. Median frequencies of IFNgamma+ cells obtained after activation by PMA-ionomycin, PHA or SEA-B were 29.3%, 20.0% and 6.8% for CD4+ cells, and 67.8%, 20.6% and 6.8% for CD8+ cells. In blood samples diluted 1/5 or 1/10, and cultured in the presence of cytomegalovirus (CMV) or varicella-zoster virus (VZV), mean peak levels of 2.8% and 1.4% IFNgamma+CD4+ cells were recorded at 120 h. The levels of cells producing cytokines other than IFNgamma were generally much lower and, in the case of IL-4 and IL-13, difficult to distinguish from background levels recorded in cultures with medium only. Kinetic studies of cytokine production by PBMCs showed a pattern similar to that of whole blood with peak levels of IFNgamma-producing cells recorded at 72 h. The increased levels of IFNgamma production after culture for 72 h were due to an expansion of the numbers of cytokine-producing cells responsive to a specific stimulus. Antigen-specific cells are usually present only at low levels in peripheral blood and may not be detected following simple activation for a few hours. To reach a level of detection in such cases, culture of diluted blood for several days is recommended.

BackgroundIntradermal 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.MethodsIn 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.Results129 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.ConclusionsA 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 registrationWorld Health Organization International Clinical Trials Registry Platform PACTR2010050002122368.

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.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.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.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.

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; P0.01). 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.