Vaccine Efficacy Studies Research Articles

Safety and serologic response to a Haemonchus contortus vaccine in alpacas

Haemonchosis in camelids remains a challenging disease to treat, and prevention has become increasingly problematic due to widespread anthelmintic resistance. Barbervax®is an adjuvanted vaccine containing natural H-11, H-gal-GP antigens obtained from Haemonchus contortus adults via a proprietary process and solubilized in Quil A. This vaccine is approved for use in Australia, after demonstrating its safety and efficacy in sheep and goats. There are no published studies evaluating Barbervax in other ruminants/pseudoruminants such as camelids which can be parasitized with H. contortus. The vaccine utilizes a mixture of the parasite gut mucosal membrane enzymes including H-gal-GP and H11, involved in digesting a blood meal from the host. This study monitored the safety profile of the Barbervax® vaccine in a group of adolescent alpacas. Although designed into the original study of vaccine efficacy, the experimental infection with viable H. contortus third stage larvae could not be completed due to lack of detectable significant variation of infection following experimental challenge. Twelve alpacas (158 + 15 days) were randomized to vaccination with Barbervax® or no treatment. Three doses of Barbervax® were administered at 3 week intervals and investigators involved in animal monitoring and sample collection were blinded to the groupings. Clinical pathologic parameters were evaluated 7 days before vaccination, and 1 and 2 months post-vaccination. Daily clinical observations were made and specific observations regarding the injection site and rectal temperatures were monitored in each alpaca twice daily for 1 week following vaccination. Fecal egg counts, packed cell volume, and total protein were monitored following challenge with 1500 H. contortus larvae on days 42, 46, and 50. An increase in rectal temperature for a duration of 2 days (range 2–4 days) was observed post-vaccination. Vaccinated alpacas were lethargic for 2-3 days following vaccination; however, they maintained an appetite and no visible or palpable injection site reactions were observed. Following the first vaccination, all animals maintained normal clinical pathologic parameters throughout the study period. The vaccinated animals did develop titers to the H. contortus antigen as measured by ELISA. In conclusion, the Barbervax® vaccine demonstrated safety in this small group of young, healthy alpacas, but additional studies are required to evaluate the efficacy of the vaccine under field conditions in protecting alpacas against infection with H. contortus.

Testing Pneumonia Vaccines in the Elderly: Determining a Case Definition for Pneumococcal Pneumonia in the Absence of a Gold Standard.

Clinical assessments of vaccines to prevent pneumococcal community-acquired pneumonia (CAP) require sensitive and specific case definitions, but there is no gold standard diagnostic test. To develop a new case definition suitable for vaccine efficacy studies, we applied latent class analysis (LCA) to the results from 7 diagnostic tests for pneumococcal etiology on clinical specimens from 323 elderly persons with radiologically confirmed pneumonia enrolled in the Finnish Community-Acquired Pneumonia Epidemiology study during 2005–2007. Compared with the conventional use of LCA, which is mainly to determine sensitivities and specificities of different tests, we instead used LCA as an appropriate instrument to predict the probability of pneumococcal etiology for each CAP case based on individual test profiles, and we used the predictions to minimize the sample size that would be needed for a vaccine efficacy trial. When compared with the conventional laboratory criteria of encapsulated pneumococci in culture, in blood culture or high-quality sputum culture, or urine antigen positivity, our optimized case definition for pneumococcal CAP resulted in a trial sample size that was almost 20,000 subjects smaller. We believe that the novel application of LCA detailed here to determine a case definition for pneumococcal CAP could also be similarly applied to other diseases without a gold standard.

Development of a new enzyme immunoassay for improved detection of rotavirus in fecal specimens of vaccinated infants

BackgroundGroup A rotavirus is the most common cause of acute diarrhea in young children worldwide. A simple and rapid enzyme immunoassay (EIA) has been commonly used to detect rotavirus infection and evaluate rotavirus vaccines. Currently licensed commercial EIA kits have low sensitivity. A more sensitive detection of rotavirus can improve rotavirus diagnostics and vaccine efficacy studies. ObjectiveA biotin-avidin based sandwich EIA was developed and compared with commercial EIA kits for improved detection of viral shedding in fecal samples from infants who received human rotavirus vaccine Rotarix in Mexico. Study designA monoclonal antibody (mAb: 1D4) specific to human rotavirus group antigen VP6 was prepared and used to develop a biotin-avidin based sandwich EIA. This EIA was employed to test 128 fecal samples from vaccinated infants, in comparison with two commercial EIA kits using RT-PCR as a reference. ResultsA new biotin-avidin based sandwich EIA showed specific reaction to group A rotaviruses, but not to other enteric viruses. This new EIA had a detection rate of 36.7% for rotavirus antigen shedding in fecal specimens, which was two times higher (16.4%, 18.0%) than those from two commercial EIA kits. ConclusionThe new EIA had specificity and higher sensitivity than commercial kits. This new EIA has the potential to detect rotavirus at lower concentration in clinical specimens and thus should be further evaluated as a more sensitive kit for use in diagnostics and vaccine efficacy and effectiveness studies.

Distinct virulence of Rift Valley fever phlebovirus strains from different genetic lineages in a mouse model.

Rift Valley fever phlebovirus (RVFV) causes high rates of abortions and fetal malformations in ruminants, and hemorrhagic fever, encephalitis, or blindness in humans. Viral transmission occurs via mosquito vectors in endemic areas, which necessitates regular vaccination of susceptible livestock animals to prevent the RVF outbreaks. Although ZH501 strain has been used as a challenge strain for past vaccine efficacy studies, further characterization of other RVFV strains is important to optimize ruminant and nonhuman primate RVFV challenge models. This study aimed to characterize the virulence of wild-type RVFV strains belonging to different genetic lineages in outbred CD1 mice. Mice were intraperitoneally infected with 1x103 PFU of wild-type ZH501, Kenya 9800523, Kenya 90058, Saudi Arabia 200010911, OS1, OS7, SA75, Entebbe, or SA51 strains. Among them, mice infected with SA51, Entebbe, or OS7 strain showed rapid dissemination of virus in livers and peracute necrotic hepatitis at 2–3 dpi. Recombinant SA51 (rSA51) and Zinga (rZinga) strains were recovered by reverse genetics, and their virulence was also tested in CD1 mice. The rSA51 strain reproduced peracute RVF disease in mice, whereas the rZinga strain showed a similar virulence with that of rZH501 strain. This study showed that RVFV strains in different genetic lineages display distinct virulence in outbred mice. Importantly, since wild-type RVFV strains contain defective-interfering RNA or various genetic subpopulations during passage from original viral isolations, recombinant RVFV strains generated by reverse genetics will be better suitable for reproducible challenge studies for vaccine development as well as pathological studies.

Immunoglobulin G1 Allotype Influences Antibody Subclass Distribution in Response to HIV gp140 Vaccination.

Antibody subclasses exhibit extensive polymorphisms (allotypes) that could potentially impact the quality of HIV-vaccine induced B cell responses. Allotypes of immunoglobulin (Ig) G1, the most abundant serum antibody, have been shown to display altered functional properties in regard to serum half-life, Fc-receptor binding and FcRn-mediated mucosal transcytosis. To investigate the potential link between allotypic IgG1-variants and vaccine-generated humoral responses in a cohort of 14 HIV vaccine recipients, we developed a novel protocol for rapid IgG1-allotyping. We combined PCR and ELISA assays in a dual approach to determine the IgG1 allotype identity (G1m3 and/or G1m1) of trial participants, using human plasma and RNA isolated from PBMC. The IgG1-allotype distribution of our participants mirrored previously reported results for caucasoid populations. We observed elevated levels of HIV gp140-specific IgG1 and decreased IgG2 levels associated with the G1m1-allele, in contrast to G1m3 carriers. These data suggest that vaccinees homozygous for G1m1 are predisposed to develop elevated Ag-specific IgG1:IgG2 ratios compared to G1m3-carriers. This elevated IgG1:IgG2 ratio was further associated with higher FcγR-dimer engagement, a surrogate for potential antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) function. Although preliminary, these results suggest that IgG1 allotype may have a significant impact on IgG subclass distribution in response to vaccination and associated Fc-mediated effector functions. These results have important implications for ongoing HIV vaccine efficacy studies predicated on engagement of FcγR-mediated cellular functions including ADCC and ADCP, and warrant further investigation. Our novel allotyping protocol provides new tools to determine the potential impact of IgG1 allotypes on vaccine efficacy.

Marmosets (Callithrix jacchus) as a non-human primate model for evaluation of candidate dengue vaccines: induction and maintenance of specific protective immunity against challenges with clinical isolates.

Dengue virus (DENV) is one of the major infectious diseases in tropical regions and approximately half of the world population is at risk of infection. Vaccines would offer an effective control measure against this disease. We previously reported on the utility of marmosets as an animal model for studying primary and secondary dengue infections. Infected marmosets consistently develop viraemia and antibody kinetics that reflect those of patients with dengue. Thus, it is important to determine the utility of marmosets as an animal model for demonstrating vaccine efficacy. In this study, marmosets were inoculated with candidate vaccine and parent strains and challenged with a clinical DENV strain. The viraemia and antibody kinetics in these marmosets were determined. Marmosets consistently develop lower viraemia with an attenuated vaccine strain. During secondary challenge, the IgM response was delayed, whereas the IgG levels rose rapidly, indicating a secondary antibody response. The neutralizing activities against the homotypic serotype were high; all marmosets were protected against viraemia following secondary inoculation. The viraemia markers and antibody responses were consistent with those of human DENV infection and vaccinees. These results demonstrate the utility of marmosets as an animal model for the study of vaccine efficacy.

Burden of hospitalized childhood community-acquired pneumonia: A retrospective cross-sectional study in Vietnam, Malaysia, Indonesia and the Republic of Korea

ABSTRACTBackground: Few studies describe the community-acquired pneumonia (CAP) burden in children in Asia. We estimated the proportion of all CAP hospitalizations in children from nine hospitals across the Republic of Korea (high-income), Indonesia, Malaysia (middle-income), and Vietnam (low/middle-income).Methods: Over a one or two-year period, children <5 years hospitalized with CAP were identified using ICD-10 discharge codes. Cases were matched to standardized definitions of suspected (S-CAP), confirmed (C-CAP), or bacterial CAP (B-CAP) used in a pneumococcal conjugate vaccine efficacy study (COMPAS). Median total direct medical costs of CAP-related hospitalizations were calculated.Results: Vietnam (three centers): 7591 CAP episodes were identified with 4.3% (95% confidence interval 4.2;4.4) S-CAP, 3.3% (3.2;3.4) C-CAP and 1.4% (1.3;1.4) B-CAP episodes of all-cause hospitalization in children aged <5 years. The B-CAP case fatality rate (CFR) was 1.3%. Malaysia (two centers): 1027 CAP episodes were identified with 2.7% (2.6;2.9); 2.6% (2.4;2.8); 0.04% (0.04;0.1) due to S-CAP, C-CAP, and B-CAP, respectively. One child with B-CAP died. Indonesia (one center): 960 CAP episodes identified with 18.0% (17.0;19.1); 16.8% (15.8;17.9); 0.3% (0.2;0.4) due to S-CAP, C-CAP, and B-CAP, respectively. The B-CAP CFR was 20%. Korea (three centers): 3151 CAP episodes were identified with 21.1% (20.4;21.7); 11.8% (11.2;12.3); 2.4% (2.1;2.7) due to S-CAP, C-CAP, and B-CAP, respectively. There were no deaths.Costs: CAP-related hospitalization costs were highest for B-CAP episodes: 145.00 (Vietnam) to 1013.3 USD (Korea) per episode.Conclusion: CAP hospitalization causes an important health and cost burden in all four countries studied (NMRR-12-50-10793).

Refinement of the equine influenza model in the natural host: A meta-analysis to determine the benefits of individual nebulisation for experimental infection and vaccine evaluation in the face of decreased strain pathogenicity

Equine Influenza (EI) is an important respiratory disease of horses caused by H3N8 equine influenza viruses (EIV). Vaccination is a key strategy to prevent or control this disease. However, EIV undergoes continuous antigenic drift and whilst numerous EI vaccines are commercially available worldwide, an accurate evaluation of their efficacy is frequently required through clinical trials conducted in the natural host. Room nebulisation is one of the chosen methods to challenge horses during EI vaccine studies. A potential decreased pathogenicity observed with recent Florida Clade 2 (FC2) EIV isolates have increased the heterogeneity of the clinical response and virus shedding measured after infection by room nebulisation, which reduced the statistical power of studies. Our objectives were to compare clinical and virological parameters following experimental infection with several different EIV strains and to confirm that individual nebulisation is a model refinement that prevents an increase of the number of animals per group. This study is a retrospective comparison and meta-analysis of clinical and virological results collected from 9 independent EIV infection studies in the natural host. Naïve Welsh mountain ponies were experimentally infected by room or individual nebulisation with FC2 EIV strains, including A/equine/Richmond/1/07 (R/07), A/equine/East Renfrewshire/11 (ER/11), A/equine/Cambremer/1/2012 (C/12) and A/equine/Northamptonshire/1/13 (N/1/13). The retrospective meta-analysis confirmed a decreased pathogenicity of the EIV ER/11 and C/12 strains when compared with R/07. Experimental infection by individual nebulisation improved the clinical and virological parameters induced by recent FC2 strains, when compared with conventional room nebulisation. In conclusion, individual nebulisation offers a better control of the challenge dose administered and a greater homogeneity of the response measured in control animals. This in turn, helps maintain the number of animals per group to the minimum necessary required to obtain meaningful results in vaccine efficacy studies, which adheres to the 3Rs (Replacement, Reduction and Refinement) principles.

Performance of Zoster Vaccine Live (Zostavax): A Systematic Review of 12 years of Experimental and Observational Evidence

BackgroundOne in three people in the U.S. will develop herpes zoster during their life. Zoster Vaccine Live (ZVL or Zostavax™), has been licensed in the U.S. since 2006 to prevent herpes zoster. ZVL protection has been shown to wane with time and estimates of effect can be imprecise. We performed a systematic review of the duration of efficacy and effectiveness of ZVL against herpes zoster (HZ).MethodsWe systematically searched PubMed, Embase, Cochrane, and clinicaltrials.gov for vaccine efficacy or effectiveness (VE) studies of ZVL. Two authors independently screened each title and abstract, and potential VE studies were reviewed in-depth. Eligibility criteria included original data on ZVL prevention of HZ in a general population of immunocompetent recipients ≤ 60 years old. Selected articles were abstracted, independently reviewed, and discrepancies adjudicated. We attempted to locate relevant unpublished work and contacted authors for additional data, where necessary. Measures of association were illustrated on a forest plot and converted to VE (1-hazard ratio or risk ratio or odds ratio).ResultsWe screened 1302 articles; 17 underwent full text review and 8 met inclusion criteria and were abstracted for this review. Selected studies included 1 phase III randomized controlled trial, 2 quasi experimental and 5 observational studies. One experimental and 5 observational studies estimated VE during the period from vaccination up to 4 years following vaccination; estimates across studies ranged from 33%-55%. Two quasi experimental and 3 observational studies estimated VE for ≥ 4 years following vaccination; estimates ranged from 19%-40%; the median estimate was 24% (Figure). Pooled VE was not calculated due to heterogeneity in length of follow up, age distribution of study subjects, as well as adjustment for factors such as underlying medical conditions.ConclusionMost experimental and observational studies estimate VE just above 50% during the 3 years following receipt of ZVL. Beyond 3 years, ZVL protection wanes, with most studies estimating a VE of ≤24% after 4 years. Information on overall efficacy and duration of protection from ZVL will guide policy decisions regarding its use.Disclosures E. Belongia, Novavax: Investigator, Research support

Whole-Genome Sequencing and Variant Analysis of Human Papillomavirus 16 Infections.

ABSTRACTHuman papillomavirus (HPV) is a strongly conserved DNA virus, high-risk types of which can cause cervical cancer in persistent infections. The most common type found in HPV-attributable cancer is HPV16, which can be subdivided into four lineages (A to D) with different carcinogenic properties. Studies have shown HPV16 sequence diversity in different geographical areas, but only limited information is available regarding HPV16 diversity within a population, especially at the whole-genome level. We analyzed HPV16 major variant diversity and conservation in persistent infections and performed a single nucleotide polymorphism (SNP) comparison between persistent and clearing infections. Materials were obtained in the Netherlands from a cohort study with longitudinal follow-up for up to 3 years. Our analysis shows a remarkably large variant diversity in the population. Whole-genome sequences were obtained for 57 persistent and 59 clearing HPV16 infections, resulting in 109 unique variants. Interestingly, persistent infections were completely conserved through time. One reinfection event was identified where the initial and follow-up samples clustered differently. Non-A1/A2 variants seemed to clear preferentially (P = 0.02). Our analysis shows that population-wide HPV16 sequence diversity is very large. In persistent infections, the HPV16 sequence was fully conserved. Sequencing can identify HPV16 reinfections, although occurrence is rare. SNP comparison identified no strongly acting effect of the viral genome affecting HPV16 infection clearance or persistence in up to 3 years of follow-up. These findings suggest the progression of an early HPV16 infection could be host related.IMPORTANCE Human papillomavirus 16 (HPV16) is the predominant type found in cervical cancer. Progression of initial infection to cervical cancer has been linked to sequence properties; however, knowledge of variants circulating in European populations, especially with longitudinal follow-up, is limited. By sequencing a number of infections with known follow-up for up to 3 years, we gained initial insights into the genetic diversity of HPV16 and the effects of the viral genome on the persistence of infections. A SNP comparison between sequences obtained from clearing and persistent infections did not identify strongly acting DNA variations responsible for these infection outcomes. In addition, we identified an HPV16 reinfection event where sequencing of initial and follow-up samples showed different HPV16 variants. Based on conventional genotyping, this infection would incorrectly be considered a persistent HPV16 infection. In the context of vaccine efficacy and monitoring studies, such infections could potentially cause reduced reported efficacy or efficiency.

Improving vaccine trials in infectious disease emergencies.

Unprecedented global effort is under way to facilitate the testing of countermeasures in infectious disease emergencies. Better understanding of the various options for trial design is needed in advance of outbreaks, as is preliminary global agreement on the most suitable designs for the various scenarios. What would enhance the speed, validity, and ethics of clinical studies of such countermeasures? Focusing on studies of vaccine efficacy and effectiveness in emergencies, we highlight three needs: for formal randomized trials-even in most emergencies; for individually randomized trials-even in many emergencies; and for six areas of innovation in trial methodology. These needs should inform current updates of protocols and roadmaps.

Abstract 4540: MiVax: an innovative cancer neoantigen prediction system

Abstract T cell epitopes bearing tumor-specific mutations discovered using next generation sequencing stimulate T cell-mediated processes that lead to tumor regression. Although neoantigen prediction using computational methods rapidly identifies epitope candidates in the mutanome, a large proportion prove to be non-immunogenic. Innovative computational tools validated for infectious disease can be applied to enhance design of personalized cancer immunotherapies by classification of predicted epitopes according to potential for mounting a tumor-specific response. We developed the JanusMatrix algorithm that parses query sequences into MHC-facing and T cell receptor (TCR)-facing sequences and screens sequence databases to identify MHC ligands that share TCR faces with host-related proteins. A database of human protein sequences is available to identify tumor-specific epitopes that may reduce anti-tumor activity by sequences that activate regulatory T cells (Tregs) trained in the thymus on self-antigens. Similarly, tumor-specific epitope candidates are screened using databases composed of human commensal- or pathogen-derived sequences to identify epitopes that, respectively, may detrimentally or beneficially cross-react with T cells raised over the course of an individual’s immune history. We conducted retrospective analyses of cancer vaccine efficacy studies performed in mice [Kreiter et al. 2015 Nature 520, 692-696] showing that mutanome-directed vaccines effective at preventing tumor growth contained higher numbers of class I and II MHC neo-epitopes and had lower potential to cross-react with other murine proteins, as identified by the MiVax platform. Likewise, an evaluation of mutanomes derived from non-small cell lung cancer patients [Rizvi et al. 2014 Science 348, 124-128] revealed that improved clinical outcomes were observed in patients with mutanomes enriched in class I MHC neo-epitopes with TCR faces distinct from other self-epitopes. While retrospective in nature, the suite of tools used for these analyses have been extensively validated in prospective vaccine studies for infectious disease. Removal of Treg epitopes identified by JanusMatrix has led to the development of a novel H7N9 influenza vaccine scheduled for Phase I clinical trial. These results highlight the benefits of using in silico tools for the selection of high-value neoantigen candidates and how they can improve the design and efficacy of cancer vaccines. Neoantigens with low Treg activation potential may then be used to support development of personalized therapies including vaccination and in vitro expansion of tumor infiltrating lymphocytes for adoptive cell transfer. Citation Format: Lenny Moise, Guilhem Richard, Frances Terry, William Martin, Ann De Groot. MiVax: an innovative cancer neoantigen prediction system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4540. doi:10.1158/1538-7445.AM2017-4540

Age of Acquiring Causal Human Papillomavirus (HPV) Infections: Leveraging Simulation Models to Explore the Natural History of HPV-induced Cervical Cancer.

Although new human papillomavirus (HPV) infections can occur at all ages, the age at which women acquire their "causal" HPV infection that develops into cervical cancer is poorly understood and practically unobservable. We aimed to estimate the age distribution at which individuals acquired their causal HPV infection in the absence of HPV vaccination or screening to help guide the optimal use of both. Using an empirically calibrated mathematical model that simulates the natural history of cervical cancer, we estimated the cumulative number of causal HPV infections by age, stratified by HPV genotype (HPV16 vs. other HPV genotypes), and the direct age-specific reduction in cancer incidence for alternative vaccination initiation scenarios (i.e., age 9-45 years). Our model projected that among all cervical cancers, 50% and 75% of women acquired their causal HPV infection by ages 20.6 (range: 20.1-21.1) and 30.6 (range: 29.6-31.6) years, respectively. HPV16 infections were acquired at an earlier age. Assuming 95% efficacy against HPV16 and HPV18 infections, the direct reduction in lifetime risk of cervical cancer varied from 55% (53-56%) among women vaccinated at age 9 years to 6% (range: 6-7%) among women vaccinated at age 45 years. Similar patterns were observed for the second-generation vaccine. Although new HPV infections and precancers can occur throughout a woman's lifetime, only a small proportion are acquired in mid-adult women and are vaccine-preventable. Our simulations highlight the potential limitations of using surrogate endpoints for vaccine efficacy studies of mid-adult women to guide policy decisions for implementation.

Rhesus macaque and mouse models for down-selecting circumsporozoite protein based malaria vaccines differ significantly in immunogenicity and functional outcomes

BackgroundNon-human primates, such as the rhesus macaques, are the preferred model for down-selecting human malaria vaccine formulations, but the rhesus model is expensive and does not allow for direct efficacy testing of human malaria vaccines. Transgenic rodent parasites expressing genes of human Plasmodium are now routinely used for efficacy studies of human malaria vaccines. Mice have however rarely predicted success in human malaria trials and there is scepticism whether mouse studies alone are sufficient to move a vaccine candidate into the clinic.MethodsA comparison of immunogenicity, fine-specificity and functional activity of two Alum-adjuvanted Plasmodium falciparum circumsporozoite protein (CSP)-based vaccines was conducted in mouse and rhesus models. One vaccine was a soluble recombinant protein (CSP) and the other was the same CSP covalently conjugated to the Qβ phage particle (Qβ-CSP).ResultsMice showed different kinetics of antibody responses and different sensitivity to the NANP-repeat and N-terminal epitopes as compared to rhesus. While mice failed to discern differences between the protective efficacy of CSP versus Qβ-CSP vaccine following direct challenge with transgenic Plasmodium berghei parasites, rhesus serum from the Qβ-CSP-vaccinated animals induced higher in vivo sporozoite neutralization activity.ConclusionsDespite some immunologic parallels between models, these data demonstrate that differences between the immune responses induced in the two models risk conflicting decisions regarding potential vaccine utility in humans. In combination with historical observations, the data presented here suggest that although murine models may be useful for some purposes, non-human primate models may be more likely to predict the human response to investigational vaccines.

Incidence of Herpes Zoster in a Large Cohort of Persons Living with HIV

Herpes Zoster (HZ) incidence has decreased over 40% in the United States but remains elevated in persons living with HIV (PLWH). HZ vaccine is not routinely recommended for PLWH and provider-prescribing patterns vary greatly. Updated incidence information in this population is needed to guide vaccine strategies. Using data from the US military HIV Natural History Study (NHS), we evaluated the incidence and risk factors for HZ in the modern ART era. NHS subjects undergo bi-annual visits with laboratory testing, examinations, and records reviewed for clinical diagnosis, including HZ. Analysis was restricted to subjects contributing to follow-up after 2001. Risk factors for HZ (demographic and HIV-specific) were assessed with a multivariate Cox proportional hazards model. Of the 2954 subjects meeting inclusion criteria, 237 (8%) were diagnosed with HZ. At HZ diagnosis, the median age, CD4 count, and viral load were 38.6 years [IQR-30.8, 45.8], 461 cells/uL [IQR-333, 638] and 1900 copies/mL [IQR-50, 19580] respectively. The incidence of HZ was highest prior to 1996 at 3.24 cases/100 person-years (PY) of follow-up (2.96–3.54) and declined significantly over time with 1.9 (1.6–2.3), 1.4 (1.2–1.8), 1.4 (1.1–1.7), and 0.9 (0.7–1.2) cases/100 PY recorded in 1996–2000, 2001–2005, 2006–2010, and 2011–2016, respectively. In the multivariate model, longer time from HIV diagnosis to ART initiation was associated with HZ. ART use, higher CD4 count, recent year of HIV diagnosis, and older age were protective (Table 1). HZ remains a common diagnosis in the ART era. NHS subjects with HZ were in the fourth and fifth decades of their life and had preserved CD4 counts highlighting the need for HZ vaccine safety and efficacy studies to help guide provider practice. Delays in ART initiation were associated with HZ emphasizing the need for compliance with current ART guidelines. Table 1. Table 1. All authors: No reported disclosures.

Abstract A018: High-value T cell epitope selection for mutanome-directed cancer immunotherapy using an innovative cancer neo-epitope classification system

Abstract T cell epitopes bearing tumor-specific mutations discovered using whole-exomic and transcriptomic sequencing of tumor-normal pairs stimulate T cell-mediated processes that lead to tumor regression. Although neo-epitope prediction using computational methods rapidly identifies epitope candidates in the mutanome, a large proportion of neo-epitopes prove to be non-immunogenic. Innovative computational tools validated for infectious disease targets can be applied to enhance design of personalized cancer immunotherapies by classification of predicted epitopes by potential for mounting a tumor-specific response. Tumor-specific epitopes with potential to stimulate naïve T cells and epitopes that may cross-react with antigen-experienced T cells raised in infection are valuable targets. Epitopes with potential to activate regulatory T cells trained on self and commensal antigens are counter-indicated. We developed the JanusMatrix algorithm that parses query sequences into MHC-facing and T cell receptor (TCR)-facing sequences and screens sequence databases to identify MHC ligands that share TCR faces with host-related proteins. A database of human protein sequences is available to identify tumor-specific epitopes that may reduce anti-tumor activity by sequences that activate regulatory T cells (Tregs) trained in the thymus on self-antigens. Similarly, tumor-specific epitope candidates are screened using databases composed of antigens derived from human commensals or pathogens to identify epitopes that, respectively, may detrimentally or beneficially cross-react with T cells raised over the course of an individual's immune history. Neo-epitope candidates are ranked according to MHC binding potential, TCR face homology. We conducted retrospective analyses of cancer vaccine efficacy studies performed in mice [Kreiter et al. 2015 Nature 520, 692–696] showing that mutanome-directed vaccines effective at preventing tumor growth contained higher numbers of class I and II MHC neo-epitopes, as identified by EpiVax's iVAX platform, and had lower potential to cross-react with other murine proteins. An evaluation of mutanomes derived from non-small cell lung cancer patients [Rizvi et al. 2014 Science 348, 124-128] confirmed these findings. Improved clinical outcomes were observed in patients with mutanomes enriched in class I MHC neo-epitopes with TCR faces distinct from other human-derived epitopes. These results highlight the benefits of using in silico prediction tools for the selection of neo-epitopes and how they can improve the design and efficacy of future cancer vaccines. While retrospective in nature, the suite of tools used for these analyses have been extensively validated in prospective vaccine studies for infectious disease. Removal of Treg epitopes, identified by JanusMatrix, has led to the development of more immunogenic vaccine antigens. EpiVax's H7N9 influenza Treg epitope-depleted vaccine is scheduled for Phase I clinical trial. Direct application of JanusMatrix to the neo-epitope-driven cancer vaccine discovery and design pipeline will focus candidate selection on higher-value sequences than what conventional T cell epitope mapping algorithms generate. Neo-epitopes with low Treg activation potential may then be used to support development of personalized therapies including vaccination and in vitro expansion of tumor infiltrating lymphocytes for adoptive cell transfer. Citation Format: Lenny Moise, Guilhem Richard, Frances Terry, William Martin, Anne De Groot. High-value T cell epitope selection for mutanome-directed cancer immunotherapy using an innovative cancer neo-epitope classification system [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A018.

Aspects of T Cell-Mediated Immunity Induced in Mice by a DNA Vaccine Based on the Dengue-NS1 Antigen after Challenge by the Intracerebral Route.

Dengue disease has emerged as a major public health issue across tropical and subtropical countries. Infections caused by dengue virus (DENV) can evolve to life-threatening forms, resulting in about 20,000 deaths every year worldwide. Several animal models have been described concerning pre-clinical stages in vaccine development against dengue, each of them presenting limitations and advantages. Among these models, a traditional approach is the inoculation of a mouse-brain adapted DENV variant in immunocompetent animals by the intracerebral (i.c.) route. Despite the historical usage and relevance of this model for vaccine testing, little is known about the mechanisms by which the protection is developed upon vaccination. To cover this topic, a DNA vaccine based on the DENV non-structural protein 1 (pcTPANS1) was considered and investigations were focused on the induced T cell-mediated immunity against i.c.-DENV infection. Immunophenotyping assays by flow cytometry revealed that immunization with pcTPANS1 promotes a sustained T cell activation in spleen of i.c.-infected mice. Moreover, we found that the downregulation of CD45RB on T cells, as an indicator of cell activation, correlated with absence of morbidity upon virus challenge. Adoptive transfer procedures supported by CFSE-labeled cell tracking showed that NS1-specific T cells induced by vaccination, proliferate and migrate to peripheral organs of infected mice, such as the liver. Additionally, in late stages of infection (from the 7th day onwards), vaccinated mice also presented reduced levels of circulating IFN-γ and IL-12p70 in comparison to non-vaccinated animals. In conclusion, this work presented new aspects about the T cell-mediated immunity concerning DNA vaccination with pcTPANS1 and the i.c. infection model. These insights can be explored in further studies of anti-dengue vaccine efficacy.

Zebrafish as a novel model for non-typhoidal Salmonella pathogenesis, transmission and vaccine efficacy

Salmonella-induced gastroenteritis causes massive morbidity and mortality in both adults and children of developing countries. However, it is difficult to study the mode of infection and vaccine efficacy due to inadequacies of current animal models. For this reason, we have explored using zebrafish as an improved model for non-typhoidal Salmonella (NTS) infection, including Salmonella enterica Typhimurium, Salmonella enterica Enteritidis and Salmonella enterica Weltevreden. In this study, we found that after infection of zebrafish with NTS, severe diarrhea like symptoms were observed and NTS significantly colonized the zebrafish intestine without any manipulation of the normal intestinal microbiota of the fish. Furthermore, these strains can colonize for longer than 72h and induce severe inflammation in the intestine, which may induce fish death. We also found that infected fish can transmit the pathogen into naïve fish. Moreover, we have established that zebrafish is an excellent model for vaccine study. Successive triple bath vaccination with heat-killed single serotype S. Typhimurium and S. Enteritidis immunogen induced protective efficacy against a high dose (108CFU/ml) of infection with these pathogens. This study provides a natural infection model for the study of NTS infection, transmission and vaccine efficacy.

A chimeric virus created by DNA shuffling of the capsid genes of different subtypes of porcine circovirus type 2 (PCV2) in the backbone of the non-pathogenic PCV1 induces protective immunity against the predominant PCV2b and the emerging PCV2d in pigs

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated disease (PCVAD). Available commercial vaccines all target PCV2a subtype, although the circulating predominant subtype worldwide is PCV2b, and the emerging PCV2d subtype is also increasingly associated with PCVAD. Here we molecularly bred genetically-divergent strains representing PCV2a, PCV2b, PCV2c, PCV2d, and “divergent PCV2a” subtypes by DNA-shuffling of the capsid genes to produce a chimeric virus representing PCV2 global genetic diversity. When placed in the PCV2a backbone, one chimeric virus (PCV2-3cl14) induced higher neutralizing antibody titers against different PCV2 subtypes. Subsequently, a candidate vaccine (PCV1-3cl14) was produced by cloning the shuffled 3cl14 capsid into the backbone of the non-pathogenic PCV1. A vaccine efficacy study revealed that chimeric virus PCV1-3cl14 induces protective immunity against challenge with PCV2b or PCV2d in pigs. The chimeric PCV1-3cl14 virus is a strong candidate for a novel vaccine in pigs infected with variable PCV2 strains.

A Single Mutation in the VP1 of Enterovirus 71 Is Responsible for Increased Virulence and Neurotropism in Adult Interferon-Deficient Mice.

Hand, foot, and mouth disease (HFMD) has spread throughout the Asia-Pacific region, affecting millions of young children, who develop symptoms ranging from painful blisters around their mouths and hands to neurological complications. Many members of the genus Enterovirus (family Picornaviridae) cause HFMD. Enterovirus 71 (EV71) is one of the primary causative agents and has been linked to severe disease. Vaccine efficacy and pathogenesis studies for EV71 have been limited because there is a lack of suitable animal models. Previously, we generated a mouse-adapted EV71 (mEV71) capable of infecting 12-week-old interferon receptor-deficient AG129 mice and used the model to evaluate the efficacy of candidate HFMD vaccines. Here, we present data investigating the genetic correlates of EV71 adaptation and characterize the virus's tissue tropism in mice. Using reverse genetics, a VP1 mutation (K244E) was shown to be necessary for mEV71 virulence in adult mice. Another VP1 mutation (H37R) was required for mEV71 recovery on rhabdomyosarcoma (RD) cells. Viral loads determined by real-time reverse transcription (RT)-PCR confirmed the presence of mEV71 in the sera and multiple organs of mice. Histological analysis revealed signs of meningitis and encephalitis, characteristic of severe human disease. The further description of this model has provided insight into EV71 pathogenesis and demonstrates the importance of the VP1 region in facilitating mEV71 adaptation. EV71 is a reemerging pathogen, and little is known about the genetic determinants involved in its pathogenesis. The absence of animal models has contributed to this lack of knowledge. The data presented here improve upon the existing animal models by characterizing a mouse-adapted strain of EV71. We determined that a VP1 mutation (K244E) was needed for EV71 virulence in adult AG129 mice. While this mutation was found previously for EV71 adaptation in 5-day-old BALB/c mice, neurotropic disease did not develop. Using interferon-deficient mice, we raised the age of susceptibility beyond 6 weeks and provided clear evidence that our model mimics severe human infections. The model can be exploited to identify determinants of EV71 virulence and to reveal molecular mechanisms that control the virus-host interaction, especially those associated with neurotropic disease. Furthermore, these data provide useful information regarding the importance of VP1, specifically position 244, in host adaptation and tissue dissemination.