Serum Responses Research Articles

Virus-like particles containing multiple antigenic proteins of Toxoplasma gondii induce memory T cell and B cell responses.

Although the efforts to develop vaccine against Toxoplasma gondii infection were made for decades, there is currently no licensed vaccine available for humans. Upon discovering a number of T or B cell epitope regions from T. gondii IMC, ROP18 and MIC8, multi-antigen VLPs or combination VLPs were generated. Mice immunized with multi-antigen VLPs or combination VLPs were challenge infected with T. gondii (ME49). T. gondii-specific IgG, IgG isotypes and IgA antibody responses, memory T and B cell responses and protection were evaluated. All the mice survived upon T. gondii challenge infection by multi-antigen VLPs vaccination. Vaccinated mice elicited higher levels of parasite-specific IgG and IgG2a antibody responses in sera, IgA antibody responses in feces, CD4+ and CD8+ T cell responses, and cytokines (IFN-γ, IL-10) responses compared to combination VLPs. In particular, the multi-antigen VLPs vaccination showed significantly higher levels of antibody secreting cell (ASC) responses, CD4+ and CD8+ effector memory T cells, and memory B cells than combination VLPs. Multi-antigen VLPs vaccination showed significant reduction of brain cyst counts and size, and all mice survived. Prediction and analysis of epitopes have indicated that IMC, ROP18 and MIC8 showed partially overlapping epitopes for T and B cells. Our results indicated that antibody responses, memory T and B cells induced by multi-antigen VLPs vaccination might contribute to the complete protection upon T. gondii (ME49) challenge infection.

Induction of systemic, mucosal and memory antibody responses targeting Vibrio cholerae O1 O-specific polysaccharide (OSP) in adults following oral vaccination with an oral killed whole cell cholera vaccine in Bangladesh.

BackgroundOral cholera vaccine (OCV) containing killed Vibrio cholerae O1 and O139 organisms (Bivalent-OCV; Biv-OCV) are playing a central role in global cholera control strategies. OCV is currently administered in a 2-dose regimen (day 0 and 14). There is a growing body of evidence that immune responses targeting the O-specific polysaccharide (OSP) of V. cholerae mediate protection against cholera. There are limited data on anti-OSP responses in recipients of Biv-OCV. We assessed serum antibody responses against O1 OSP, as well as antibody secreting cell (ASC) responses (a surrogate marker for mucosal immunity) and memory B cell responses in blood of adult recipients of Biv-OCV in Dhaka, Bangladesh.Methodology/Principal findingsWe enrolled 30 healthy adults in this study and administered two doses of OCV (Shanchol) at days 0 and 14. Blood samples were collected before vaccination (day 0) and 7 days after each vaccination (day 7 and day 21), as well as on day 44. Serum responses were largely IgA with minimal IgG and IgM responses in this population. There was no appreciable boosting following day 14 vaccination. There were significant anti-OSP IgA ASC responses on day 7 following the first vaccination, but none after the second immunization. Anti-OSP IgA memory B cell responses were detectable 30 days after completion of the vaccination series, with no evident induction of IgG memory responses. In this population, anti-Ogawa OSP responses were more prominent than anti-Inaba responses, perhaps reflecting impact of previous exposure. Serum anti-OSP responses returned to baseline within 30 days of completing the vaccine series.ConclusionOur results call into question the utility of the 2-dose regimen separated by 14 days in adults in cholera endemic areas, and also suggest that Biv-OCV-induced immune responses targeting OSP are largely IgA in this highly endemic cholera area. Studies in children in cholera-endemic areas need to be performed. Protective efficacy that extends for more than a month after vaccination presumably is mediated by direct mucosal immune response which is not assessed in this study. Our results suggest a single dose of OCV in adults in a cholera endemic zone may be sufficient to mediate at least short-term protection.

Effects of Assam tea extract on growth, skin mucus, serum immunity and disease resistance of Nile tilapia (Oreochromis niloticus) against Streptococcus agalactiae

The present study aimed to assess the possible effects of Assam tea (Camellia sinensis) extract (ATE) on growth performances, immune responses, and disease resistance of Nile tilapia, Oreochromis niloticus against Streptococcus agalactiae. Five levels of ATE were supplemented into the based diet at 0, 1, 2, 4, and 8 g kg−1 feed of Nile tilapia fingerlings (10.9 ± 0.04 g initial weight) in triplicate. After four and eight weeks of feeding, fish were sampled to determine the effects of the tea supplements upon their growth performance, as well as serum and mucosal immune responses. A disease challenge using S. agalactiae was conducted at the end of the feeding trial. Fish fed ATE revealed significantly improved serum lysozyme, peroxidase, alternative complement (ACH50), phagocytosis, and respiratory burst activities compared to the basal control fed fish (P < 0.05). The mucus lysozyme and peroxidase activities were ameliorated through ATE supplementation in the tilapia diets. Supplementation of ATE significantly (P < 0.05) enhanced final body weight, weight gain, and specific growth rate; while a decreased feed conversion ratio was revealed at 2 g kg−1 inclusion level, after four and eight weeks. Challenge test showed that the relative percent survival (RSP) of fish in each treatment was 33.33%, 60.00%, 83.33%, 76.68%, and 66.68% in groups fed 0, 1, 2, 4, and 8 g kg−1, respectively. In summary, diets supplemented with ATE especially at 2 g kg−1 increased the humoral and mucosal immunity, enhanced growth performance, and offered higher resistance against S. agalactiae infection in Nile tilapia.

Influenza Virus-Like Particles Presenting both Toxoplasma gondii ROP4 and ROP13 Enhance Protection against T. gondii Infection.

Rhoptry organelle proteins (ROPs) secreted by Toxoplasma gondii (T. gondii) play a critical role during parasite invasion into host cells. In this study, virus-like particles (VLPs) vaccines containing ROP4 and/or ROP13 together with influenza M1 were generated. ROP4+ROP13 VLPs were produced by combining ROP4 VLPs with ROP13 VLPs, and ROP(4 + 13) VLPs by co-infecting insect cells with recombinant baculovirus expressing ROP4 or ROP13. Mice intranasally immunized with ROP(4 + 13) VLPs showed significantly higher levels of IgG, IgG1, IgG2a and IgA antibody responses in sera compared to ROP4+ROP13VLPs. Upon challenge infection by oral route, mice immunized with ROP(4 + 13) VLPs elicited higher levels of IgG and IgA antibody responses in fecal, urine, intestine and vaginal samples as well as CD4+ T, CD8+ T cells, and germinal center B cell responses compared to other type of vaccines, ROP4 VLPs, ROP13 VLPs, and ROP4+ROP13 VLPs. ROP(4 + 13) VLPs vaccination showed a significant decrease in the size and number of cyst in the brain and less body weight loss compared to combination ROP4+ROP13 VLPs upon challenge infection with T. gondii ME49. These results indicated that the ROP(4 + 13) VLPs vaccination provided enhanced protection against T. gondii infection compared to ROP4+ROP13 VLPs, providing an important insight into vaccine design strategy for T. gondii VLPs vaccines.

Immune Profile in Blood Following Non-convulsive Epileptic Seizures in Rats.

Non-convulsive status epilepticus (NCSE) is a prolonged epileptic seizure with subtle symptoms that may delay clinical diagnosis. Emerging experimental evidence shows brain pathology and epilepsy development following NCSE. New diagnostic/prognostic tools are therefore needed for earlier and better stratification of treatment. Here we examined whether NCSE initiates a peripheral immune response in blood serum from rats that experienced electrically-induced NCSE. ELISA analysis showed an acute transient increase in serum protein levels including interleukin-6 6 h post-NCSE, similar to the immune reaction in the brain. At 4 weeks post-NCSE, when 75% of rats subjected to NCSE had also developed spontaneous seizures, several immune proteins were altered. In particular, markers associated with microglia, macrophages and antigen presenting cells, such as CD68, MHCII, and galectin-3, were increased and the T-cell marker CD4 was decreased in serum compared to both non-stimulated controls and NCSE rats without spontaneous seizures, without correlation to interictal epileptiform activity. Analyses of serum following intracerebral injection of lipopolysaccharide (LPS) showed an acute increase in interleukin-6, but at 4 weeks unaltered levels of MHCII and galectin-3, an increase in CD8 and CD11b and a decrease in CD68. None of the increased serum protein levels after NCSE or LPS could be confirmed in spleen tissue. Our data identifies the possibility to detect peripheral changes in serum protein levels following NCSE, which may be related to the development of subsequent spontaneous seizures.

Neuroinflammation caused by mental stress: the effect of chronic restraint stress and acute repeated social defeat stress in mice

ABSTRACTObjectives: Cerebrovascular disease (CVD) is the leading cause of permanent disability worldwide. Inflammation has been reported to play an important role in the progression of CVD. Neuropsychiatric disorders such as depression are associated with increased incidence of CVD epidemiologically, although the mechanisms underlying this association are not clear. In this study, we assessed the effect of the acute repeated social defeat stress (RSDS) and chronic restraint stress (CRS) on neuroinflammation in mice.Methods: A total of 40 6-week-old male C57BL/6J mice were divided into RSDS, CRS, and corresponding control groups. In the RSDS group, male C57BL/6J mice were repeatedly subjected to bouts of social defeat by a larger CD-1 mouse for 10 min daily for 10 consecutive days. In the CRS group, the mice were exposed to restraint stress for 6 h per day for 28 consecutive days. Depressive behavior was evaluated by conducting sucrose preference test over 24 h. Peripheral blood serum and brain tissues were collected for measurement of corticosterone (CORT), epinephrine (EPI), and inflammatory factors (TNF-α and IL-6) using ELISA or real-time PCR 24 h after the sucrose preference test.Results: Both RSDS and CRS decreased the sucrose preference ratio. The acute stress increased serum CORT and EPI, while the chronic stress did not significantly influence them. Both stress models induced an inflammatory response in peripheral serum and the brain.Conclusions: RSDS and CRS are two effective models of depressive behavior, and both models cause neuroinflammation, which may be responsible for the increased risk of CVD seen in patients with depression.

HIV-1/SIV Humoral Responses in External Secretions

The protective function of mucosal HIV-1- or SIV-specific antibodies against viral infection has stimulated extensive studies of their Ig isotype association with differences in specificity and in effector functions. In contrast to many mucosally acquired microbial infections in which the humoral responses are dominated by induction of secretory IgA (S-IgA), HIV-1/SIV infections stimulate vigorous IgG responses in sera as well as in external secretions but low IgA virus-specific antibodies although the total levels of IgA in these fluids remain unaltered. The diminished or even absent IgA responses to HIV-1/SIV and to other mucosal antigens in external secretions and their replacement with IgG is likely to influence the functionality of mucosal barriers and eliminate antiinflammatory effector functions of IgA antibodies. Furthermore, the polymeric character of S-IgA with 4-8 antigen-binding sites, exquisite resistance to proteolysis and anti-inflammatory potential are of great advantage in mucosal protection. The markedly different effector functions of mucosal antibodies of IgG and IgA isotypes must be considered in the design of HIV-1 vaccines to stimulate S-IgA responses at sites of virus entry and IgG responses in the systemic compartment.

Early-Stage Alzheimer's Disease Is Associated with Simultaneous Systemic and Central Nervous System Dysregulation of Insulin-Linked Metabolic Pathways.

Brain insulin resistance is a well-recognized abnormality in Alzheimer's disease (AD) and the likely mediator of impaired glucose utilization that emerges early and progresses with disease severity. Moreover, the rates of mild cognitive impairment (MCI) or AD are significantly greater in people with diabetes mellitus or obesity. This study was designed to determine whether systemic and central nervous system (CNS) insulin resistant disease states emerge together and thus may be integrally related. Insulin-related molecules were measured in paired human serum and cerebrospinal fluid (CSF) samples from 19 with MCI or early AD, and 21 controls using a multiplex ELISA platform. In MCI/AD, both the CSF and serum samples had significantly elevated mean levels of C-peptide and an incretin, and reduced expression of Visfatin, whereas only CSF showed significant reductions in insulin and leptin and only serum had increased glucagon, PAI-1, and ghrelin. Although the overall CSF and serum responses reflected insulin resistance together with insulin deficiency, the specific alterations measured in CSF and serum were different. In MCI and early-stage AD, CNS and systemic insulin-related metabolic dysfunctions, including insulin resistance, occur simultaneously, suggesting that they are integrally related and possibly mediated similar pathogenic factors.

Salmonella-vectored vaccine delivering three Clostridium perfringens antigens protects poultry against necrotic enteritis.

Necrotic enteritis is an economically important poultry disease caused by the bacterium Clostridium perfringens. There are currently no necrotic enteritis vaccines commercially available for use in broiler birds, the most important target population. Salmonella-vectored vaccines represent a convenient and effective option for controlling this disease. We used a single attenuated Salmonella vaccine strain, engineered to lyse within the host, to deliver up to three C. perfringens antigens. Two of the antigens were toxoids, based on C. perfringens α-toxin and NetB toxin. The third antigen was fructose-1,6-bisphosphate aldolase (Fba), a metabolic enzyme with an unknown role in virulence. Oral immunization with a single Salmonella vaccine strain producing either Fba, α-toxoid and NetB toxoid, or all three antigens, was immunogenic, inducing serum, cellular and mucosal responses against Salmonella and the vectored C. perfringens antigens. All three vaccine strains were partially protective against virulent C. perfringens challenge. The strains delivering Fba only or all three antigens provided the best protection. We also demonstrate that both toxins and Fba are present on the C. perfringens cell surface. The presence of Fba on the cell surface suggests that Fba may function as an adhesin.

Abstract B032: PhIP-seq assessment of the serum antibody repertoire before and after immune-related adverse events in four melanoma patients treated with checkpoint blockade immunotherapy

Abstract Phage immunoprecipitation sequencing (PhIP-seq) is a technique to profile the epitope specificities of an antibody repertoire by phage display of a peptide library followed by immunoprecipitation and next-generation sequencing (1). We have developed phage libraries corresponding to all 36-mer peptides in the human proteome (approximately 413,000 peptides), as well as libraries of peptides found in viruses, bacteria, and toxins. Here, in a pilot study to understand if PhIP-seq might be used to identify predictive biomarkers for immune-related adverse events (irAE) in the context of checkpoint blockade immunotherapy, we applied the human proteome library to probe the self-directed IgG response in sera from four melanoma patients receiving checkpoint blockade who experienced irAE. In total, 16 serum samples acquired at the pre-treatment, post-treatment / pre-irAE, or post-irAE timepoints were assayed from these patients. We additionally analyzed samples from six melanoma patients who received vaccines targeting the MART-1 and NY-ESO-1 antigens. Using a stringent confidence threshold, we identified a median 31 self-directed antibody specificities (hits) at the pre-immunotherapy timepoint in these cancer patients and 41.5 hits post-therapy, compared to a median 6.5 hits in healthy donors. Patients receiving combination checkpoint blockade showed more hits than those treated with monotherapy. For a patient who received combination nivolumab and ipilimumab and developed a hepatic irAE, a cluster of 25 hits (of 161 total) was detected uniquely in a sample taken within two months subsequent to the adverse event. This cluster included a peptide from the C-Reactive Protein (CRP) gene and other genes expressed in the liver (EHBP1, VSTM2L), or across tissues (SAFB2), but also included genes with low expression in the liver (SPTBN4, DMBT1, IQGAP3, SPTBN4). In a patient who received ipilimumab and nivolumab and developed myalgia, hits against targets associated with autoimmune disease were found including NUMA1 (connective tissue autoimmunity), TRPM1 (melanoma-associated retinopathy), as well as three epitopes in the Mediator Of DNA Damage Checkpoint 1 (MDC1) gene. Finally, for three patients receiving a peptide vaccine targeting MART-1 and NY-ESO-1 with adjuvant poly-ICLC and montanide, we detected an IgG response against NY-ESO-1, but not MART-1, in each patient. No response to either protein was observed in three patients receiving a dendritic cell vaccine against the same antigens.Our small study suggests that PhIP-seq readily detects changes in the epitope specificities of the serum antibody repertoire in the course of immunotherapy. A key limitation of PhIP-seq is that the method can only detect antibodies with specificities for linear (as opposed to conformational) epitopes. Nevertheless, the assay’s low cost (approximately $30 per sample), small sample size requirement (1 µL serum), and ability to work with dried blood spot samples may make it an attractive technology to discover serological predictors of irAE in larger cohorts.

Association of HLA-dependent islet autoimmunity with systemic antibody responses to intestinal commensal bacteria in children.

Microbiome sequence analyses have suggested that changes in gut bacterial composition are associated with autoimmune disease in humans and animal models. However, little is known of the mechanisms through which the gut microbiota influences autoimmune responses to distant tissues. Here, we evaluated systemic antibody responses against cultured human gut bacterial strains to determine whether observed patterns of anticommensal antibody (ACAb) responses are associated with type 1 diabetes (T1D) in two cohorts of pediatric study participants. In the first cohort, ACAb responses in sera collected from participants within 6 months of T1D diagnosis were compared with age-matched healthy controls and also with patients with recent onset Crohn's disease. ACAb responses against multiple bacterial species discriminated among these three groups. In the second cohort, we asked whether ACAb responses present before diagnosis were associated with later T1D development and with HLA genotype in participants who were discordant for subsequent progression to diabetes. Serum IgG2 antibodies against Roseburia faecis and against a bacterial consortium were associated with future T1D diagnosis in an HLA DR3/DR4 haplotype-dependent manner. These analyses reveal associations between antibody responses to intestinal microbes and HLA-DR genotype and islet autoantibody specificity and with a future diagnosis of T1D. Further, we present a platform to investigate antibacterial antibodies in biological fluids that is applicable to studies of autoimmune diseases and responses to therapeutic interventions.

Abstract B113: Protective serum responses by CRISPR/Cas9-edited primary B cells expressing antibodies of choice

Abstract Attempts to create an effective, traditional vaccine against several pathogens such as HIV have so far failed and current research suggests that in the case of HIV protective serum responses mediated by broadly-neutralizing antibodies (bNAbs) will only be achieved by multiple, sequential immunizations. Identifying, producing and implementing a regimen consisting of a series of immunogens that lead to a relatively consistent, protective immune response in a heterogeneous population will be highly challenging. We are developing an alternative approach in which autologous primary B cells are edited to express an antibody of choice, in this case a bNAb against HIV. Following adoptive transfer the individual is immunized with a cognate antigen to activate the edited B cells and generate a protective, humoral immune response to HIV. This method can be adapted for any combination of high-affinity antibody and antigen. We have developed a CRISPR/Cas9-based method to target the endogenous IgH and IgK loci in mice. Both loci must be targeted in order to avoid producing B cells with chimeric and potentially self-reactive receptors. In addition, both of the bNAb encoding chains must be coordinately expressed. To do so we assemble an RNP consisting of Cas9 and single stranded DNA (ssDNA) encoding IgK followed by a P2A self-cleaving oligopeptide and the VDJ exon. A separate Cas9/guide complex is included to ablate the endogenous IgK locus. Since the heavy chain variable region is knocked into the endogenous locus in between the VDJ and constant regions normal isotype switching can occur and only on-target integrations can express the full BCR of choice. B cell survival is dependent on Ig expression and therefore all of the B cells that have lost Igk expression and do not properly integrate the bNAb will die. Using this approach, we have created cells carrying a synthetic intermediate of the anti-HIV bNAb 3BNC60. Transfer of 3BNC60 synthetic intermediate-edited cells into congenically marked wild-type mice followed by immunization with a cognate Env-derived antigen showed that the edited cells were able to start an immune response and create 3BNC60 synthetic intermediate serum antibody with the predicted epitope specificity of different isotypes. One of the potential benefits to this approach is that it would not be limited to a single monoclonal. B cells could be edited to express different antibodies and then be transferred together. Our initial experiments show that the approach is feasible but the process must be optimized to become practical; particularly, the number of correctly edited B cells needs to be increased for efficient transfer. We are also working on translating these experiments from mice to macaques in order to do protection experiments.We believe the approach presented here would have wide-ranging implications for both research and public health. It boasts several advantages compared to traditional vaccination, particularly in the context of HIV as it skips bNAb evolution, can potentially combine more than one type of bNAb and, as opposed to a sequential immunization, could potentially be achieved in a two-visit procedure (blood draw followed by injections). Since the approach uses primary B cells that are modified in their endogenous loci, which then mature into plasma cells in vivo, and not other cell types ectopically expressing an antibody, the immune response should in principle be boostable, long-lived and allow for bNAb diversification by somatic hypermutation. As opposed to an immunogen series in a sequential immunization, our approach is not limited to a particular bNAb and relies only on a functional bNAb and antigen combination as opposed to an entire series of antigens. Lastly, this approach is not limited to HIV and could be applicable to other diseases in which constant antibody intervention is indicated. Citation Format: Harald Hartweger, Mila Jankovic, Michel C. Nussenzweig. Protective serum responses by CRISPR/Cas9-edited primary B cells expressing antibodies of choice [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B113.

Immunization of young heifers with staphylococcal immune evasion proteins before natural exposure to Staphylococcus aureus induces a humoral immune response in serum and milk

BackgroundStaphylococcus aureus, a leading cause of mastitis in dairy cattle, causes severe mastitis and/or chronic persistent infections with detrimental effects on the cows’ wellbeing, lifespan and milk production. Despite years of research there is no effective vaccine against S. aureus mastitis. Boosting of non-protective pre-existing immunity to S. aureus, induced by natural exposure to S. aureus, by vaccination may interfere with vaccine efficacy. The aim was to assess whether experimental immunization of S. aureus naïve animals results in an immune response that differs from immunity following natural exposure to S. aureus.ResultsFirst, to define the period during which calves are immunologically naïve for S. aureus, Efb, LukM, and whole-cell S. aureus specific serum antibodies were measured in a cohort of newborn calves by ELISA. Rising S. aureus specific antibodies indicated that from week 12 onward calves mounted an immune response to S. aureus due to natural exposure. Next, an experimental immunization trial was set up using 8-week-old heifer calves (n = 16), half of which were immunized with the immune evasion molecules Efb and LukM. Immunization was repeated after one year and before parturition and humoral and cellular immunity specific for Efb and LukM was determined throughout the study. Post-partum, antibody levels against LukM and EfB were significantly higher in serum, colostrum and milk in the experimentally immunized animals compared to animals naturally exposed to S. aureus. LukM specific IL17a responses were also significantly higher in the immunized cows post-partum.ConclusionsExperimental immunization with staphylococcal immune evasion molecules starting before natural exposure resulted in significantly higher antibody levels against Efb and LukM around parturition in serum as well as the site of infection, i.e. in colostrum and milk, compared to natural exposure to S. aureus. This study showed that it is practically feasible to vaccinate S. aureus naïve cattle and that experimental immunization induced a humoral immune response that differed from that after natural exposure only.

Specific immune response in mucosal and systemic compartments of Cirrhinus mrigala vaccinated against Edwardsiella tarda: In vivo kinetics using different antigen delivery routes

Mucosal immune barriers confer protection against invading fish pathogens. Here, we conducted an experiment for 60 days to assess the mucosal and systemic immune response in Mrigal (Cirrhinus mrigala), an Indian major carp. Fish were immunized with inactivated Edwardsiella tarda by four different routes, namely, oral, immersion, injection, and anal intubation. An indirect enzyme‐linked immunosorbent assay (ELISA) was used to measure the specific immune response (antibody) in serum and mucus (collected from skin, gill, and gut) of the fish on 0, 15, 30, 45, and 60 days postimmunization. For specific immune response in the serum, significantly higher (p &lt; 0.05) optical density (OD) values were obtained in the anal group (0.52 ± 0.03) and in the oral group (0.48 ± 0.03). In the skin mucus, significantly higher OD values were obtained in the oral group (0.48 ± 0.04) and immersion group (0.32 ± 0.03). In the gill mucus, significantly higher OD values were obtained in the oral group (0.82 ± 0.08) and the immersion group (0.73 ± 0.03). In the gut mucus, significantly higher OD values were obtained in the immersion group (0.080 ± 0.007) compared to the rest of the treatments. Fish from all the groups were challenged with LD50 dose of E. tarda at the end of the experiment. We conclude that oral and immersion immunization routes offer better protection of C. mrigala compared to other antigen delivery routes.

Induction of protective immunity by recombinant peptidoglycan associated lipoprotein (rPAL) protein of Legionella pneumophila in a BALB/c mouse model

Legionella pneumophila causes a severe form of pneumonia known as Legionnaires’ disease especially in patients with impaired cellular immune response. In order to prevent the disease, immunogenicity and the level of the induction of protective immunity from the recombinant peptidoglycan–associated lipoprotein (rPAL) against Legionella pneumophila in BALB/c mice was examined. Mice immunized with (rPAL) rapidly increased an antibody response in serum and also displayed a strong activation of both innate and adaptive cell-mediated immunity as determined by antigen-specific splenocyte proliferation, an early production of pro-inflammatory cytokines in the serum and in the splenocyte cultures. Infection with a primary sublethal does of Legionella pneumophila serogroup 1, strain paris, caused resistance to a lethal challenge infection in the animals with 100% survival rate. However, mice treated with rPAL survived with 60% rate in 10 days after a lethal i.v challenge with L. pneumophila. All of the control animals receiving PBS died within 24 h. The present study indicates that recombinant protein PAL of Legionella pneumophila is strongly immunogenic and capable to elicit early innate and adaptive immune responses and lasting immunity against a lethal dose of Legionella pneumophila challenge. Antigenic characterization and immune protection of recombinant protein PAL would be of considerable value in comprehension the immune-pathogenesis of the disease and in development possible vaccine against the Legionella.

The Effect of Preexisting Immunity on Virus Detection and Immune Responses in a Phase II, Randomized Trial of a Russian-Backbone, Live, Attenuated Influenza Vaccine in Bangladeshi Children

BackgroundIn a 2012 Phase II clinical trial, 300 Bangladeshi children aged 24 to 59 months with no prior influenza vaccine exposure were randomized to receive a single intranasally-administered dose of either trivalent, Russian-backbone, live, attenuated influenza vaccine (LAIV) or placebo. Protocol-defined analyses, presented in the companion manuscript, demonstrate decreased viral detection and immunogenicity for A/H1N1pdm09, relative to the A/H3N2 and B strains. This post hoc analysis of the trial data aims to investigate the LAIV strain differences by testing the hypothesis that preexisting humoral and mucosal immunity may influence viral recovery and immune responses after LAIV receipt.MethodsWe used logistic regressions to evaluate the relations between markers of preexisting immunity (ie, hemagglutination inhibition [HAI], microneutralization, and immunoglobulin G and immunoglobulin A (both serum and mucosal antibodies) and LAIV viral recovery in the week post-vaccination. We then tested for potential effect modification by baseline HAI titers (ie, <10 versus ≥10) and week 1 viral recovery on the LAIV-induced serum and mucosal immune responses, measured between days 0 and 21 post-vaccination.ResultsHigher levels of preexisting immunity to influenza A/H3N2 and B were strongly associated with strain-specific prevention of viral shedding upon LAIV receipt. While evidence of LAIV immunogenicity was observed for all 3 strains, the magnitudes of immune responses were most pronounced in children with no evidence of preexisting HAI and in those with detectable virus.ConclusionsThe results provide evidence for a bidirectional association between viral replication and immunity, and underscore the importance of accounting for preexisting immunity when evaluating virologic and immunologic responses to LAIVs.Clinical Trials RegistrationNCT01625689.

1970. Phase 1 Clinical Trial of Intranasal Immunization with M2-Deficient, Single Replication, Live Influenza Vaccine (M2SR): Safety and Immune Response in Adults

BackgroundInfluenza vaccines are needed with greater effectiveness and breadth of coverage. FluGen is developing M2SR (M2 deficient Single Replication), an investigational, live virus vaccine. M2SR contains the internal proteins of donor A/Puerto Rico/8/34 and hemagglutinin (HA) and neuraminidase (NA) selected from targeted Type A influenza strains. M2SR undergoes only a single round of infection in the respiratory epithelium but evokes an immune response profile similar to wild-type influenza viruses. In influenza naïve and pre-immune ferrets, M2SR protects against multiple influenza A subtypes.MethodsA Phase 1, first-in-human, randomized, placebo-control study (FluGen-H3N2-V001; ClinicalTrials.gov identifier NCT02822105) was conducted at a single USA site, with 96 adults, ages 18–49 years. Study vaccine contained HA and NA from A/Brisbane/10/2007 (H3N2). Study volunteers received a single intranasal (IN) inoculation with either M2SR at dose levels of 106, 107 or 108 TCID50 or saline placebo (N = 24/cohort). Study subjects were evaluated for virus replication and solicited local and systemic reactions for 7 days, all adverse events (AE) for 28 days and serious AE (SAE) for 180 days.ResultsNo infectious virus was detected in nasal swabs from any vaccinated subject. The most commonly reported AE was mild nasal rhinorrhea/congestion during the first 7 days after vaccination (Figure 1). No subject had fever or a severe reaction to the vaccine. No SAEs were reported. At least one AE was reported among 29%, 58%, and 83% of M2SR subjects administered 106, 107, or 108 TCID50, respectively, and 46% among placebo subjects. There were no notable imbalances among study groups for other events. T- and B-cell responses, including influenza-specific serum and mucosal antibody responses were detected at a significantly higher frequency among vaccine than placebo subjects (Figure 2).Figure 1.Common adverse events first 7 days post-vaccinationFigure 2.Influenza-specific immune responsesConclusionM2SR vaccine was safe and well tolerated at all dose levels, generated a dose-response effect for humoral (HA antibody) and mucosal antibodies against both homologous and heterologous influenza variants, and elicited robust T-cell responses. No infectious virus was detected in nasal swabs from any vaccinated subject.Disclosures J. Eiden, FluGen, Inc.: Consultant, Consulting fee and Stock Options. G. Gordon, FluGen: Consultant, Consulting fee. C. Fierro, FluGen: Investigator, Fee for service. R. Belshe, FluGen: Consultant, Consulting fee and Stock Options. H. Greenberg, FluGen: Consultant, Consulting fee and Stock OpTions. D. Hoft, FluGen: Research Contractor, Fee for service. Y. Hatta, FluGen: Employee, Salary and Stock Options. M. Imboden, FluGen: Employee, Salary and Stock Options. M. Moser, FluGen: Employee, Salary and Stock Options. R. Herber, FluGen: Employee, Salary and Stock Options. D. Boltz, FluGen: Research Contractor, fee for service. M. Tary-Lehmann, FluGen: Research Contractor, Fee for service. CTL: Founder and Owner, Salary. Y. Kawaoka, FluGen: Founder, Stock Options. G. Neumann, FluGen: Founder, Stock Options. P. Radspinner, FluGen: Board Member and Founder, Salary and Stock Options. R. Aitchison, FluGen: Consultant, Consulting fee. P. Bilsel, FluGen: Employee, Salary and Stock Options.

Immune Correlates of Protection Induced by Virus-Like Particles Containing 2009 H1N1 Pandemic Influenza HA, NA or M1 Proteins

ABSTRACTInfluenza virus-like particle (VLPs) vaccines are a promising alternative to conventional egg-based vaccines. Evaluation of vaccine efficacy induced by HA-M1 VLPs, NA-M1 VLPs or M1 VLPs against virus challenge infection would provide important insight into vaccine design strategy. In this study, we generated VLPs containing hemagglutinin (HA), neuraminidase (NA) or M1 proteins derived from the A/California/04/09. Mice were immunized intramuscularly with HA-M1, NA-M1 or M1 VLPs and protective immunity was evaluated by assessing lung virus loads against low (5LD50) or high (100LD50) lethal dose of homologous virus challenges. High levels of virus-specific serum IgG antibody responses were induced in mice after HA-M1 VLPs immunization, whereas low or no IgG antibody responses were detected from immunization with NA-M1 VLPs or M1 VLPs, independently. Mice that were immunized with HA-M1 VLPs showed below the limit of detection on lung virus loads against low dose (5LD50) of challenge and significant reduction against high dose (100LD50) of challenge infection. Mice that were immunized with NA-M1 or M1 VLPs also displayed reduced lung viral loads compared to naïve control. In vitro cultures of cells from mouse spleen and bone marrow revealed that HA-M1 VLPs and NA-M1 VLPs induced higher levels of antibody-secreting cell (ASC) responses compared to naïve control, whereas M1 VLPs showed no ASC responses. HA-M1, NA-M1 or M1 VLPs immunization demonstrated varying degree of protection with respect to body weight changes and survival rates, which are consistent with the levels of antibody responses in sera and ASC responses from spleen and bone marrow.

Characterization of immunogenicity of avian influenza antigens encapsulated in PLGA nanoparticles following mucosal and subcutaneous delivery in chickens.

Mucosal vaccine delivery systems have paramount importance for the induction of mucosal antibody responses. Two studies were conducted to evaluate immunogenicity of inactivated AIV antigens encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs). In the first study, seven groups of specific pathogen free (SPF) layer-type chickens were immunized subcutaneously at 7-days of age with different vaccine formulations followed by booster vaccinations two weeks later. Immune responses were profiled by measuring antibody (Ab) responses in sera and lachrymal secretions of vaccinated chickens. The results indicated that inactivated AIV and CpG ODN co-encapsulated in PLGA NPs (2x NanoAI+CpG) produced higher amounts of hemagglutination inhibiting antibodies compared to a group vaccinated with non-adjuvanted AIV encapsulated in PLGA NPs (NanoAI). The tested adjuvanted NPs-based vaccine (2x NanoAI+CpG) resulted in higher IgG responses in the sera and lachrymal secretions at weeks 3, 4 and 5 post-vaccination when immunized subcutaneously. The incorporation of CpG ODN led to an increase in Ab-mediated responses and was found useful to be included both in the prime and booster vaccinations. In the second study, the ability of chitosan and mannan coated PLGA NPs that encapsulated AIV and CpG ODN was evaluated for inducing antibody responses when delivered via nasal and ocular routes in one-week-old SPF layer-type chickens. These PLGA NPs-based and surface modified formulations induced robust AIV-specific antibody responses in sera and lachrymal secretions. Chitosan coated PLGA NPs resulted in the production of large quantities of lachrymal IgA and IgG compared to mannan coated NPs, which also induced detectable amounts of IgA in addition to the induction of IgG in lachrymal secretions. In both mucosal and subcutaneous vaccination approaches, although NPs delivery enhanced Ab-mediated immunity, one booster vaccination was required to generate significant amount of Abs. These results highlight the potential of NPs-based AIV antigens for promoting the induction of both systemic and mucosal immune responses against respiratory pathogens.

140P - Oral cavity immune response in pancreatic ductal adenocarcinoma (PDAC)

Background: We studied the humoral immune response in patients with pancreatic ductal adenocarcinoma (PDAC) using mouthwashes from the oral cavity. We found lower immune response against recall antigens, fusobacterium nucleatum and tumor associated antigens before immunotherapy treatment. We also found better prognosis when the antibodies against tested antigens got higher. Finally, we found significant differences in the humoral immune response in the oral cavity against serum. Methods: Antigens from fusobacterium nucleatum, recall and tumor associated antigens were tested by IgG + IgM + IgA ELISA in both oral cavity and serum of PDAC patients n = 50. We provided 5 ml of saline solution to all the patients and controls to rinse the oral cavity hardly for 5 minutes and deposit the content in 15 ml tube. Sera were obtained. All was done after the approval of the local IRB committee. Results: All the evaluated antigens were lower in the oral cavity of PDAC patients compared with controls (p = 0.001). Serum responses for all antigens were significantly different than oral cavity (p = 0.03). At the end of the immunotherapy treatment consisting in a combination of immunomodulation, multi peptide antigen specific active immunotherapy and immunogenic chemotherapy with doxorubicin and oxaliplatin the levels of antibodies were increased significantly. For example, FAP (p = 0.0001), EGFR (p = 0.005), Fascin-1 (p = 0001), VCP (P = 001). Also, the immunotherapy combination significantly increased antibody immune response against fusobaterium lysate (p = 0001). Conclusions: PDAC is a challenge disease especially in late stages. PDAC patients had low antibody titters against several antigens in comparison with controls and before treatment the oral cavity. Serum antibodies were lower than oral cavity even after treatment. Oral cavity antibodies may be a useful early biomarker in patients in high risk of PDAC. This data also suggest that antibodies get low when the cancer or premalignant lesions of PDAC are present. Legal entity responsible for the study: Centro de Investigacion de Cancer en Sonora (CICS). Funding: CICS USA. Disclosure: All authors have declared no conflicts of interest.