Equivalence of ELISpot Assays Demonstrated between Major HIV Network Laboratories

The Comprehensive T Cell Vaccine Immune Monitoring Consortium (CTC-VIMC) was created to provide standardized immunogenicity monitoring services for HIV vaccine trials. The ex vivo interferon-gamma (IFN-γ) ELISpot is used extensively as a primary immunogenicity assay to assess T cell-based vaccine candidates in trials for infectious diseases and cancer. Two independent, GCLP-accredited central laboratories of CTC-VIMC routinely use their own standard operating procedures (SOPs) for ELISpot within two major networks of HIV vaccine trials. Studies are imperatively needed to assess the comparability of ELISpot measurements across laboratories to benefit optimal advancement of vaccine candidates.We describe an equivalence study of the two independently qualified IFN-g ELISpot SOPs. The study design, data collection and subsequent analysis were managed by independent statisticians to avoid subjectivity. The equivalence of both response rates and positivity calls to a given stimulus was assessed based on pre-specified acceptance criteria derived from a separate pilot study.Detection of positive responses was found to be equivalent between both laboratories. The 95% C.I. on the difference in response rates, for CMV (-1.5%, 1.5%) and CEF (-0.4%, 7.8%) responses, were both contained in the pre-specified equivalence margin of interval [-15%, 15%]. The lower bound of the 95% C.I. on the proportion of concordant positivity calls for CMV (97.2%) and CEF (89.5%) were both greater than the pre-specified margin of 70%. A third CTC-VIMC central laboratory already using one of the two SOPs also showed comparability when tested in a smaller sub-study.The described study procedure provides a prototypical example for the comparison of bioanalytical methods in HIV vaccine and other disease fields. This study also provides valuable and unprecedented information for future vaccine candidate evaluations on the comparison and pooling of ELISpot results generated by the CTC-VIMC central core laboratories

Characterization of neutrophil subsets in healthy human pregnancies.

We have previously shown that in successful pregnancies increased arginase activity is a mechanism that contributes to the suppression of the maternal immune system. We identified the main type of arginase-expressing cells as a population of activated low-density granulocytes (LDGs) in peripheral blood mononuclear cells and in term placentae. In the present study, we analyzed the phenotype of LDGs and compared it to the phenotype of normal density granulocytes (NDGs) in maternal peripheral blood, placental biopsies and cord blood. Our data reveal that only LDGs but no NDGs could be detected in placental biopsies. Phenotypically, NDGs and LDGs from both maternal and cord blood expressed different levels of maturation, activation and degranulation markers. NDGs from the maternal and cord blood were phenotypically similar, while maternal, cord and placental LDGs showed different expression levels of CD66b. LDGs present in cord blood expressed higher levels of arginase compared to maternal and placental LDGs. In summary, our results show that in maternal and cord blood, two phenotypically different populations of neutrophils can be identified, whereas in term placentae, only activated neutrophils are present

Percent of Arginase1⁺CD15⁺ LDGs present in placentae, cord blood and maternal blood.

<p>LDGs were isolated from maternal and cord blood and placentae (n = 7) as described in materials and methods. The percentage of CD15⁺ arginase⁺ cells was determined by flow cytometry. Statistical significance was determined by a Kruskal-Wallis test.</p

Comparison of expression levels of arginase and phenotypic markers of LDGs in placentae, cord and maternal blood.

<p>LDGs were isolated from maternal and cord blood (n = 7) as described in materials and methods. Expression levels (MFI) of phenotypic markers were determined by flow cytometry. Statistical significance was determined by a two-tailed Mann-Whitney test.</p

Expression levels of arginase and phenotypic markers of NDGs and LDGs in maternal peripheral blood.

<p>NDGs and LDGs were isolated from maternal blood (n = 7) as described in materials and methods. Expression levels (MFI) of phenotypic markers were determined by flow cytometry. Statistical significance was determined by a two-tailed Mann-Whitney test.</p

Comparison of the phenotype of LDGs in neonate and maternal blood and placentae.

<p>LDGs were isolated as described in materials and methods (n = 7) and the expression levels of CD66b was determined by flow cytometry. Statistical significance was determined by a kruskal-Wallis test. Box = interquartile range and median; whiskers = range.</p

Percentage of LDGs in neonatal and maternal blood and placentae.

<p>LDGs were isolated as described in materials and methods (n = 7) and the percentage of CD15⁺ arginase⁺ cells was determined by flow cytometry. The percentage of LDGs present in the peripheral blood of healthy controls was 0.24±0.3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085696#pone.0085696-Cloke1" target="_blank">[12]</a> Statistical significance was determined by a kruskal-Wallis test. Box = interquartile range and median; whiskers = range.</p

Phenotypic analysis of LDGs and NDGs.

<p>LDGs and NDGs were isolated as described in materials and methods (n = 7) and the expression levels of arginase, CD66b, CD15, CD63, CD33 and CD16 were determined by flow cytometry. Statistical significance was determined by a two-tailed Mann-Whitney test. Box = interquartile range and median; whiskers = range.</p

Expression levels of arginase and phenotypic markers of NDGs and LDGs in cord blood.

<p>NDGs and LDGs were isolated from cord blood (n = 7) as described in materials and methods. Expression levels (MFI) of phenotypic markers were determined by flow cytometry. Statistical significance was determined by a two-tailed Mann-Whitney test.</p

Immune activation alters cellular and humoral responses to yellow fever 17D vaccine

This study aimed to evaluate the contribution of the patient-specific immune microenvironment to the response to the licensed yellow fever vaccine 17D (YF-17D) in an African cohort.Background. Defining the parameters that modulate vaccine responses in African populations will be imperative to design effective vaccines for protection against HIV, malaria, tuberculosis, and dengue virus infections. This study aimed to evaluate the contribution of the patient-specific immune microenvironment to the response to the licensed yellow fever vaccine 17D (YF-17D) in an African cohort. Methods. We compared responses to YF-17D in 50 volunteers in Entebbe, Uganda, and 50 volunteers in Lausanne, Switzerland. We measured the CD8+ T cell and B cell responses induced by YF-17D and correlated them with immune parameters analyzed by flow cytometry prior to vaccination. Results. We showed that YF-17D–induced CD8+ T cell and B cell responses were substantially lower in immunized individuals from Entebbe compared with immunized individuals from Lausanne. The impaired vaccine response in the Entebbe cohort associated with reduced YF-17D replication. Prior to vaccination, we observed higher frequencies of exhausted and activated NK cells, differentiated T and B cell subsets and proinflammatory monocytes, suggesting an activated immune microenvironment in the Entebbe volunteers. Interestingly, activation of CD8+ T cells and B cells as well as proinflammatory monocytes at baseline negatively correlated with YF-17D–neutralizing antibody titers after vaccination. Additionally, memory T and B cell responses in preimmunized volunteers exhibited reduced persistence in the Entebbe cohort but were boosted by a second vaccination. Conclusion. Together, these results demonstrate that an activated immune microenvironment prior to vaccination impedes efficacy of the YF-17D vaccine in an African cohort and suggest that vaccine regimens may need to be boosted in African populations to achieve efficient immunity. Trial registration. Registration is not required for observational studies. Funding. This study was funded by Canada’s Global Health Research Initiative, Defense Threat Reduction Agency, National Institute of Allergy and Infectious Diseases, Bill & Melinda Gates Foundation, and United States Agency for International Development