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The recent spread of Zika virus (ZIKV) through the Americas and Caribbean and its devastating consequences for pregnant women and their babies have driven the search for a safe and efficacious ZIKV vaccine. Among the vaccine candidates, a first-generation ZIKV purified inactivated vaccine (ZPIV), adjuvanted with aluminum hydroxide, developed by the Walter Reed Army Institute of Research (WRAIR), has elicited high seroconversion rates in participants in three phase-I clinical trials. In collaboration with the WRAIR, Sanofi Pasteur (SP) optimized the production scale, culture and purification conditions, and increased the regulatory compliance, both of which are critical for clinical development and licensure of this vaccine. Using a clinical batch of the first-generation ZPIV as a benchmark, we report that different doses of the optimized vaccine (ZPIV-SP) elicited sustained neutralizing antibodies, specific T- and memory B-cells, and provided complete protection against a ZIKV challenge in cynomolgus macaques. These data provide evidence that the ZPIV-SP vaccine performs at least as well as the ZPIV vaccine, and provide support for continued development in the event of future ZIKV outbreaks.

Rationale: Lung-resident memory T cells (TRM) play a critical role in protecting against respiratory infection. Maintained in a quiescent state, TRM are poised for rapid effector function upon reactivation with cognate antigen. Kronoscan is a clinic-ready, fibre-based, optical imaging system developed by the University of Edinburgh, capable of measuring both fluorescence intensity and lifetime. The aim of this study is to develop a staining protocol to enable the detection of human and non-human primate (NHP) pulmonary TRM populations in situ. Methods: Human lungs (deemed non-suitable for transplantation) following ex vivo lung perfusion (EVLP) and NHP lungs (cynomolgus macaque obtained from Charles River Laboratories) were enzymatically digested to achieve a single cell suspension. Cells were stained with cross-reactive antibodies prior to flow cytometric analysis. Fluorescence intensity and lifetime imaging was also performed using Kronoscan on MACs-enriched, antibody-stained, pulmonary T cells. Results: Human and NHP TRM were primarily identified using CD69 and CD103 and co-expressed a number of chemokine receptors (CXCR3, CCR10), adhesion molecules (CD49a, CD49d) and the immune checkpoint inhibitor PD-1. Differences in phenotype were observed between CD4+ and CD8+ TRM. TRM could be identified via fluorescence intensity and lifetime imaging using Kronoscan. Conclusions: A cross-reactive antibody panel capable of detecting human and NHP pulmonary TRM was successfully developed. Fibre-based fluorescence lifetime imaging may provide a valuable tool for assessing pulmonary TRM populations in situ, reflecting the level of immunity following infection and vaccination.

After decades of inconsequential infections, and sporadic outbreaks in the Asia-Pacific region between 2007 and 2013, Zika virus caused a widespread epidemic in South America in 2015 that was complicated by severe congenital infections. After the WHO declared a Public Health Emergency of International Concern in February 2016, vaccine development efforts based on different platforms were initiated. Several candidates have since been evaluated in clinical phase I studies. Of these, a Zika purified inactivated vaccine (ZPIV), adjuvanted with aluminum hydroxide, developed by the Walter Reed Army Institute of Research (WRAIR), yielded high seroconversion rates. Sanofi Pasteur further optimized the vaccine in terms of production scale, purification conditions and regulatory compliance, using its experience in flavivirus vaccine development. Here we report that the resulting optimized vaccine (ZPIV-SP) elicited robust seroneutralizing antibody responses and provided complete protection from homologous Zika virus strain challenge in immunocompetent BALB/c mice. ZPIV-SP also showed improved immunogenicity compared with the first-generation vaccine, and improved efficacy in the more permissive interferon receptor-deficient A129 mice. Finally, analysis of the IgG response directed towards nonstructural protein 1 (NS1) suggests that viral NS1 was efficiently removed during the optimized purification process of ZPIV-SP. Together, these results suggest that the optimized vaccine is well suited for further evaluation in larger animal models and late-stage clinical studies.

International audience; Intradermal (ID) vaccination constitutes a promising approach to induce anti-infectious immunity. This route of immunization has mostly been studied with influenza split-virion vaccines. However, the efficacy of ID vaccination for sub-unit vaccines in relation to underlying skin innate immunity remains to be explored for wider application in humans. Relevant animal models that more closely mimic human skin immunity than the widely used mouse models are therefore necessary. Here, we show in domestic swine, which shares striking anatomic and functional properties with human skin, that a single ID delivery of pseudorabies virus (PRV) glycoproteins without added adjuvant is sufficient to trigger adaptive cellular and humoral immune responses, and to confer protection from a lethal respiratory infection with PRV. Analysis of early events at the skin injection site revealed up-regulation of pro-inflammatory cytokine and chemokine genes, recruitment of neutrophils and monocytes and accumulation of inflammatory DC. We further show that the sustained induction of pro-inflammatory cytokine genes results from the combined effects of skin puncture, liquid injection in the dermis and viral antigens. These data highlight that immune protection against respiratory infection can be induced by ID vaccination with a subunit vaccine and reveal that adjuvant requirements are circumvented by the mechanical and antigenic stress caused by ID injection, which triggers innate immunity and mobilization of inflammatory DC at the immunization site. ID vaccination with sub-unit vaccines may thus represent a safe and efficient solution for protection against respiratory infections in swine and possibly also in humans, given the similarity of skin structure and function in both species.

Recent data obtained with the live-attenuated tetravalent dengue CYD-TDV vaccine showed higher protective efficacy against dengue virus type 4 (DENV-4) than against DENV-2. In contrast, results from previous studies in nonhuman primates predicted comparable high levels of protection against each serotype. Maximum viral loads achieved in macaques by subcutaneous inoculation of DENV are generally much lower than those observed in naturally dengue virus-infected humans. This may contribute to an overestimation of vaccine efficacy. Using more-stringent DENV infection conditions consisting of the intravenous inoculation of 10 7 50% cell culture infectious doses (CCID 50 ) in CYD-TDV-vaccinated macaques, complete protection (i.e., undetectable viral RNA) was achieved in all 6 monkeys challenged with DENV-4 and in 6/18 of those challenged with DENV-2, including transiently positive animals. All other infected macaques (12/18) developed sustained DENV-2 RNAemia (defined as detection of viral RNA in serum samples) although 1 to 3 log 10 units below the levels achieved in control animals. Similar results were obtained with macaques immunized with either CYD-TDV or monovalent (MV) CYD-2. This suggests that partial protection against DENV-2 was mediated mainly by CYD-2 and not by the other CYDs. Postchallenge induction of strong anamnestic responses, suggesting efficient vaccine priming, likely contributed to the reduction of DENV-2 RNAemia. Finally, an inverse correlation between DENV RNA titers postchallenge and vaccine-induced homotypic neutralizing antibody titers prechallenge was found, emphasizing the key role of these antibodies in controlling DENV infection. Collectively, these data show better agreement with reported data on CYD-TDV clinical vaccine efficacy against DENV-2 and DENV-4. Despite inherent limitations of the nonhuman primate model, these results reinforce its value in assessing the efficacy of dengue vaccines. IMPORTANCE The nonhuman primate (NHP) model is the most widely recognized tool for assessing the protective activity of dengue vaccine candidates, based on the prevention of postinfection DENV viremia. However, its use has been questioned after the recent CYD vaccine phase III trials, in which moderate protective efficacy against DENV-2 was reported, despite full protection against DENV-2 viremia previously being demonstrated in CYD-vaccinated monkeys. Using a reverse translational approach, we show here that the NHP model can be improved to achieve DENV-2 protection levels that show better agreement with clinical efficacy. With this new model, we demonstrate that the injection of the CYD-2 component of the vaccine, in either a monovalent or a tetravalent formulation, is able to reduce DENV-2 viremia in all immunized animals, and we provide clear statistical evidence that DENV-2-neutralizing antibodies are able to reduce viremia in a dose-dependent manner.

A tuberculosis (TB) vaccine consisting of a recombinant fusion protein (H4) and a novel TLR9 adjuvant (IC31) is in clinical development. To better understand the H4-IC31 ratio, we measured the binding capacity of IC31 for H4 protein and immunized mice with formulations that contained limiting to excess ratios of IC31 to H4. An immunomodulated H4-specific IFNγ response was only observed when IC31 was present in excess of H4. Since TLR expression is species-specific and the vaccine is intended to boost BCG-primed immunity, we questioned whether data in mice would translate to humans. To address this question, we used the fresh human Whole Blood (hWB) recovered from BCG-vaccinated subjects to screen H4-IC31 formulations. We found IC31 modulation in hWB to be quite distinct from the TLR4-Adjuvant. Unlike TLR4-Adjuvant, IC31 formulations did not induce the pro-inflammatory cytokine TNFα, but modulated a robust H4-specific IFNγ response after 12 d of culture. We then re-stimulated the fresh hWB of 5 BCG-primed subjects with formulations that had excess or limiting IC31 binding for H4 protein and again found that an immunomodulated H4-specific IFNγ response needed an excess of IC31. Finally, we monitored the zeta (ζ) potential of H4-IC31 formulations and found that the overall charge of H4-IC31 particles changes from negative to positive once IC31 is in greater than 9-fold excess. Using two diverse yet mutually supportive approaches, we confirm the need for an excess of IC31 adjuvant in H4 TB vaccine formulations and suggest surface potential may be an important factor.

Background Two large-scale efficacy studies with the recombinant yellow fever-17D–dengue virus, live-attenuated, tetravalent dengue vaccine (CYD-TDV) candidate undertaken in Asia (NCT01373281) and Latin America (NCT01374516) demonstrated significant protection against dengue disease during two years’ active surveillance (active phase). Long-term follow up of participants for breakthrough disease leading to hospitalization is currently ongoing (hospital phase). Methodology/Principal findings We assessed the cytokine profile in acute sera from selected participants hospitalized (including during the active phase) up to the beginning of the second year of long-term follow up for both studies. The serum concentrations of 38 cytokines were measured in duplicate using the Milliplex Human Cytokine MAGNETIC BEAD Premixed 38 Plex commercial kit (Millipore, Billerica, MA, USA). Partial least squares discriminant analyses did not reveal any difference in the overall cytokine profile of CYD-TDV and placebo recipients hospitalized for breakthrough dengue regardless of stratification used. In addition, there was no difference in the cytokine profile for breakthrough dengue among those aged, Author Summary A live-attenuated, tetravalent dengue vaccine (CYD-TDV) has been shown to provide protection against dengue disease in two large-scale, placebo-controlled, phase III efficacy studies. Continued surveillance of study participants was subsequently undertaken to better define longer term vaccine efficacy and safety. A yet unexplained higher incidence of hospitalization for dengue disease was observed among children aged

Intanza® 9 μg (Sanofi Pasteur), a trivalent split-virion vaccine administered by intradermal (ID) injection, was approved in Europe in 2009 for the prevention of seasonal influenza in adults 18 to 59 years. Here, we examined the immune responses induced in adults by the ID 9 μg vaccine and the standard trivalent intramuscular (IM) vaccine (Vaxigrip® 15 μg, Sanofi Pasteur). This trial was a randomized, controlled, single-center, open-label study in healthy adults 18 to 40 years of age during the 2007/8 influenza season. Subjects received a single vaccination with the ID 9 μg (n = 38) or IM 15 μg (n = 42) vaccine. Serum, saliva, and peripheral blood mononuclear cells were collected up to 180 days post-vaccination. Geometric mean hemagglutination inhibition titers, seroprotection rates, seroconversion rates, and pre-vaccination-to-post-vaccination ratios of geometric mean hemagglutination inhibition titers did not differ between the two vaccines. Compared with pre-vaccination, the vaccines induced similar increases in vaccine-specific circulating B cells at day 7 but did not induce significant increases in vaccine-specific memory B cells at day 180. Cell-mediated immunity to all three vaccine strains, measured in peripheral blood mononuclear cells, was high at baseline and not increased by either vaccine. Neither vaccine induced a mucosal immune response. These results show that the humoral and cellular immune responses to the ID 9 μg vaccine are similar to those to the standard IM 15 μg vaccine.

The third variable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein gp120 is a major target of neutralizing antibodies in infected persons and in experimental immunized animals. Given the high degree of sequence variability of V3, the humoral response toward this region is very type-specific. In the present study, we evaluated the potential of a single peptide and an anti-idiotypic antibody to broaden the anti-V3 antibody specificity in BALB/c mice. We show that a synthetic peptide derived from the V3 determinant of HIV-1 MN isolate (V3MN), when used as an immunogen, was able to induce an antibody response to multiple (up to six) HIV-1 strains. The extent of this cross-reactivity, which tended to enlarge as the injections increased, appeared to be inversely correlated with the binding affinity to V3MN peptide. These data thus present evidence that, despite its great sequence heterogeneity, the V3 loop encompasses conserved amino-acid positions and/or stretches which may be less immunogenic than their variable counterparts. We additionally demonstrate that a rabbit anti-idiotype (Ab2), recognizing a binding site related idiotype on a V3-specific mouse monoclonal antibody (Ab1), could mount a broadened humoral response (Ab3) in mice. Unlike nominal antibody Ab1 which strictly reacted with the European HIV-1 LAI isolate, elicited Ab3 recognized the two divergent HIV-1 strains SF2 and 1286, originating respectively from North America and Central Africa, in addition to LAI. The reasons accounting for this Ab2-induced enlargement of the V3 antibody response are discussed. Our findings suggest that single peptide and anti-idiotype based immunizations may provide viable approaches to overcome, at least in part, HIV epitope variability.

Two human IgGk monoclonal antibodies (HuMAbs), termed 48-16 and 50-61A, were derived by Epstein-Barr virus transformation of B cells from two HIV-1-infected donors. These HuMAbs recognized discrete, nonoverlapping, and conformational or discontinuous epitopes on the gp120 envelope protein of HIV-1. The binding affinities of 48-16 and 50-61A for recombinant gp120 from HIV-1LAI strain, reflected by their dissociation constants, were estimated to be 2-5 x 10(-9) and 2.4 x 10(-10) M, respectively. 48-16 was shown to react with a conserved determinant present on a variety of divergent laboratory isolates, residing outside the CD4-binding site and the V3 region, which remains to be determined. 48-16 did not display, however, any detectable functional activity. 50-61A exhibited a more restricted recognition pattern, but was able to completely inhibit the 2 HIV-1 laboratory strains LAI and SF2 in a concentration range of 0.5-10 micrograms/ml, as measured by an antigen capture assay. The ability of 50-61A to block the interaction between recombinant gp120LAI and recombinant as well as cellular CD4 indicated that 50-61A epitope was localized near or within the CD4-binding side. We also demonstrated that 50-61A- and 48-16-defined epitopes (or closely related epitopes) were immunogenic in infected humans, since serum samples from 45 seropositive subjects were able to inhibit both gp120LAI-HuMAb recognitions. However, the presence of "50-61A-like" antibodies in these sera could not be associated with their neutralizing activities of HIV-1LAI infection. Interest in producing such antibodies for characterization of the human B cell repertoire to HIV-1 and their potential use in passive immunotherapy or vaccination strategy against AIDS are discussed.

We tested the potential of anti-idiotypic antibodies to function as molecular mimics of a neutralizing epitope of the human immunodeficiency virus (HIV). Three monoclonal antibodies to the third variable domain (V3) of gp120 from the HIV-1LAI isolate were raised in a BALB/c mouse. They bound gp120LAI with high affinity (K congruent to 10(-11) M), immunoprecipitated viral gp120LAI, recognized HIVLAI-infected cells by immunofluorescence and neutralized HIVLAI infection in vitro. Mice and rabbits were immunized against each one of these antibodies, and one monoclonal and two polyclonal anti-idiotypic reagents were selected for further study. The three anti-idiotypes recognized binding site-related idiotopes that were not present on other V3-specific mouse sera or monoclonal antibodies. Groups of mice and rabbits were immunized against these anti-idiotypes presented in a homopolymerized form, conjugated to a protein carrier, or in an uncoupled form. In the absence of antigen, mice that received polyclonal anti-idiotypes mounted an antibody response to gp120LAI, but not to V3-deleted gp120LAI recombinant proteins. Unlike the three nominal mAbs, the induced antibodies also reacted with gp120 from the divergent HIV strain SF2. The isotypic distribution of the anti-idiotype-induced antibodies and their temporal evolution resemble the restricted pattern and the rapid rise of the antibody response obtained after administration of recombinant gp120LAI to mice. These data thus present evidence that anti-idiotypes raised against HIV-specific antibodies may substitute viral antigens for induction of a humoral immune response to HIV. They also suggest that the HIV epitope variability may be, in part, overcome with the use of anti-idiotypic reagents.

Several motifs have been found to be the target of the neutralizing antibody response to HIV, the human immunodeficiency virus. One of the well characterized motifs maps to a loop within the third hypervariable region (V3) of the exterior envelope glycoprotein gp120 at amino acid positions 308-331 and is referred to as the principal neutralizing determinant (PND). The sequence of this V3 loop raises the question of the immunogenicity and the degree of diversity of the antibody response to the PND. We show here that this neutralization-related motif is highly immunogenic in HIV-positive subjects and in experimentally immunized primates and rodents submitted to various anti-HIV immunization regimens. In probing the diversity of the antibody response to PNDs corresponding to 11 HIV sequence-divergent isolates in serum samples of 101 HIV-positive individuals we found that human antibodies exhibit binding affinity to up to nine PND synthetic peptides. This antibody binding was in all cases tested inhibitable by the homologous PND synthetic peptide. We additionally demonstrate that this antibody cross-reactivity towards sequence-divergent PNDs is detectable in the sera of mice and chimpanzees experimentally immunized against a single HIV-1 isolate. Finally, we noticed that there is a hierarchy of reactivity among the various PNDs wherein the synthetic peptide corresponding to the MN isolate was generally the most prominently recognized by antibodies of human, non-human primate, and rodent origins. Based on these findings and on features of the sequences analyzed we suggest that, despite its overall sequence variability, the PND encompasses conserved amino acid positions or epitopes that are the targets of antibodies recognizing sequence-divergent isolates. We also propose that the high positive charge density of the most frequently recognized PNDs and the high antigenicity value of some of their residues are critical to the broad immunoreactivity of this neutralization-related motif.

Pandemic influenza vaccines have been manufactured using the A/California/07/2009 (H1N1) strain as recommended by the World Health Organization. We evaluated in mice the immunogenicity of pandemic (H1N1) 2009 vaccine and the impact of prior vaccination against seasonal trivalent influenza vaccines (TIV) on antibody responses against pandemic (H1N1) 2009. In naïve mice, a single dose of unadjuvanted H1N1 vaccine (3 microg of HA) was shown to elicit hemagglutination inhibition (HI) antibody titers40, a titer associated with protection in humans against seasonal influenza. A second vaccine dose of pandemic (H1N1) 2009 vaccine strongly increased these titers, which were consistently higher in mice previously primed with TIV than in naïve mice. At a low immunization dose (0.3 microg of HA), the AF03-adjuvanted vaccine elicited higher HI antibody titers than the corresponding unadjuvanted vaccines in both naïve and TIV-primed animals, suggesting a potential for antigen dose-sparing. These results are in accordance with the use in humans of a split-virion inactivated pandemic (H1N1) 2009 vaccine formulated with or without AF03 adjuvant to protect children and young adults against influenza A (H1N1) 2009 infection.

Detection of peptide-specific antibodies by the conventional ELISA technique is sometimes hampered by the difficulties encountered in immobilizing stretches of amino acids on the solid support. To improve the attachment of synthetic peptides to the solid phase, we have developed a sensitive and rapid immunoassay based on the irradiation of polystyrene plates with UV light prior to coating the target peptide. This pretreatment increases the specific signal in a dose-dependent manner without augmenting the background or altering the specificity of the assay. This simple method was shown to be suitable for the quantitation of murine monoclonal antibodies as well as human and rabbit polyclonal antibodies. It should be applicable to a variety of synthetic peptides and polystyrene ELISA plates. Using this technique, we were able to localize the antigenic motifs recognized by neutralizing monoclonal antibodies generated against the envelope protein gp120 of the human immunodeficiency virus.

Poxviruses are large DNA viruses capable of infecting a broad range of animal species. Infection is generally accompanied by an inflammatory response in the host, the extent of which varies considerably with the specific poxvirus and host species. Regarding ALVAC, a poxvirus derived from the canarypox vaccine strain, Kanapox, and which represents a promising immunization vehicle in humans, nothing is known about its inflammatory capacity. The present study was aimed at documenting this issue in rodents, including mice and guinea pigs. It was then attempted to evaluate how such properties could influence the immunogenicity of an antigen concomitantly administered with ALVAC preparations using the HIV envelope subunit, rgp160, as the model immunogen. The results revealed that ALVAC, either infectious or heat-inactivated, induced in both animal species an early inflammatory response, as evidenced by a rapid migration of neutrophils to the site of inoculation. In parallel, the canarypoxvirus was shown to strongly adjuvant the co-administered immunogen, resulting in a marked increase in Env-specific IgG, IgG1 and particularly IgG2(a) serum titers. Of further interest, the heat-inactivated preparation of ALVAC retained this immunostimulatory activity. Whether or not a link between the inflammatory and immunomodulatory properties of ALVAC exists remains to be established, but such features are clearly interesting with respect to the potential use of ALVAC as an immunization vehicle.

Vaccination by intramuscular injection of naked DNA is very efficient in the mouse, but immunogenicity of DNA vaccines needs to be improved in man. The aim of our study was to determine in BALB/c mice if suitable electric pulses delivered to the muscle after DNA injection – a procedure called electrotransfer — could improve the immunogenicity of suboptimal doses of a DNA vaccine expressing the influenza hemagglutinin protein. The results show a significant enhancement of the cellular and antibody responses following electrotransfer for the 1- and 10-μg DNA doses, respectively, but no effect on a lower dose. At the 10-μg dose, the IgG and hemagglutination inhibition mean titres were increased 25-fold and the inter-individual variability was markedly reduced.

Programmed-cell death (apoptosis) is a physiological cell death process which appears exacerbated in peripheral lymphocytes from HIV-infected persons. On the contrary, a barely detectable level of apoptosis is found in peripheral lymphocytes from HIV-infected chimpanzees, which support long-term productive infection without developing AIDS. In the present study, we analyzed the relationship between apoptosis and the general state of immune activation in PBMC from HIV-infected humans and chimpanzees. In addition, apoptosis control in the CD8 subset by the bcl-2 proto-oncogene was compared in both human and chimpanzees. Taken together, the results indicate that the degree of apoptosis correlates with the state of activation of the immune system and this observation together with the finding that apoptosis concerns all lymphocyte subsets indicates that the low level of apoptosis in HIV-infected chimpanzees is related to the lack of immune activation in this nonpathogenic model.

Production of cross-reactive antibodies recognizing the V3 loop--that is, the principal neutralizing determinant (PND)--of various HIV-1 isolates is an important challenge in the development of passive immunotherapy or vaccinations against AIDS. We have produced two types of antibodies to the V3 domain of HIV-1: (1) antibodies against the HIV-1 MN laboratory strain generated in rabbits and (2) antibodies targeted to the HIV-1 LAI laboratory strain induced in chimpanzees. These antibodies were shown to be specific for HIV-1 subtype B. The cross-reactivity of these antibodies has been evaluated against a large panel of peptides representing different parts of the V3 loop. Seventy-five peptides, referred to as clinical peptides, were synthesized according to HIV-1 sequences recovered from PMBCs of 27 patients followed in three Parisian hospitals. Thirteen V3 peptides derived from 4 HIV-1 laboratory strains (MN, LAI, SF2, and RF) were also included in the study. The results show that both the amino-terminal and central parts of the V3 loop are immunogenic. The rabbit antibodies against the amino-terminal end of the PND proved to be highly cross-reactive against the clinical peptides. The anti-gp160 antibodies induced in one chimpanzee recognized a significant proportion of the panel of V3 clinical sequences. These antibodies cross-reacted mainly with the apex of the V3 loop. These data give some additional indications on the immunogenicity of the V3 loop and further demonstrate that extensive cross-reactivity of anti-V3 antibodies can be obtained on field HIV-1 isolates despite the high variability of the V3 loop amino acid sequence.