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Both preexisting immunity to influenza and age have been shown to be correlates of influenza vaccine responses. Frailty, an indicator of functional impairment in older adults, was also shown in one study to predict lower influenza vaccine responses among nonveterans. In the current study, we aimed to determine the associations between frailty, preexisting immunity, and immune responses to influenza vaccine among older veterans. We studied 117 subjects (age range, 62 to 95 years [median age, 81 years]), divided into three cohorts based on the Fried frailty test, i.e., nonfrail (NF) ( n = 23 [median age, 68 years]), prefrail ( n = 50 [median age, 80 years]), and frail ( n = 44 [median age, 82 years]), during the 2010-2011 and 2011-2012 influenza seasons. Subjects received the seasonal trivalent inactivated influenza vaccine, and baseline and postvaccination samples were obtained. Anti-influenza humoral immunity, as measured by hemagglutination inhibition (HI) and microneutralization assays, was measured for influenza B, A(H1N1)pdm09, and A(H3N2) viruses. Postvaccination titers were not different between frail and NF subjects overall in this older subset of veterans. However, preexisting HI titers were strongly correlated with postvaccination titers among all functional status groups. When microneutralization titers were compared, the association between preexisting immunity and vaccine responses varied by frailty status, with the strongest correlation being observed for the NF group. In conclusion, preexisting immunity rather than frailty appeared to predict postvaccination titers in this older veteran cohort., (Copyright © 2017 American Society for Microbiology.)

Both live attenuated influenza vaccines (LAIV) and inactivated influenza vaccines (IIV) induce protective immunity against influenza. There is evidence that LAIV induces superior protection in children, whereas IIV may induce superior protection in adults. The immune mechanisms responsible for these differences have not been identified. We previously compared LAIV and IIV in young children of 6 to 36 months of age, and we demonstrated that while both induced similar hemagglutination inhibition (HAI) antibody responses, only LAIV induced significant increases in T cell responses. In the present study, 37 healthy adult subjects of 18 to 49 years of age were randomized to receive seasonal influenza vaccination with LAIV or IIV. Influenza virus-specific HAI, T cell, and secretory IgA (sIgA) responses were studied pre- and postvaccination. In contrast to the responses seen in young children, LAIV induced only minimal increases in serum HAI responses in adults, which were significantly lower than the responses induced by IIV. Both LAIV and IIV similarly induced only transient T cell responses to replication-competent whole virus in adults. In contrast, influenza virus-specific sIgA responses were induced more strongly by LAIV than by IIV. Our previous studies suggest that LAIV may be more protective than IIV in young children not previously exposed to influenza virus or influenza vaccines due to increased vaccine-induced T cell and/or sIgA responses. Our current work suggests that in adults with extensive and partially cross-reactive preexisting influenza immunity, LAIV boosting of sIgA responses to hemagglutinin (HA) and non-HA antigenic targets expressed by circulating influenza virus strains may be an important additional mechanism of vaccine-induced immunity., (Copyright © 2017 American Society for Microbiology.)

Human influenza A(H3N2) viruses that predominated during the moderately severe 2014-2015 influenza season differed antigenically from the vaccine component, resulting in reduced vaccine effectiveness (VE). To examine antibody responses to 2014-2015 inactivated influenza vaccine (IIV) and live-attenuated influenza vaccine (LAIV) among children and adolescents, we collected sera before and after vaccination from 150 children aged 3 to 17 years enrolled at health care facilities. Hemagglutination inhibition (HI) assays were used to assess the antibody responses to vaccine strains. We evaluated cross-reactive antibody responses against two representative A(H3N2) viruses that had antigenically drifted from the A(H3N2) vaccine component using microneutralization (MN) assays. Postvaccination antibody titers to drifted A(H3N2) viruses were higher following receipt of IIV (MN geometric mean titers [GMTs], 63 to 68; 38 to 45% achieved seroconversion) versus LAIV (MN GMT, 22; only 3 to 5% achieved seroconversion). In 9- to 17-year-olds, the highest MN titers were observed among IIV-vaccinated individuals who had received LAIV in the previous season. Among all IIV recipients aged 3 to 17 years, the strongest predictor of antibody responses to the drifted viruses was the prevaccination titers to the vaccine strain. The results of our study suggest that in an antigenically drifted influenza season, vaccination still induced cross-reactive antibody responses to drifted circulating A(H3N2) viruses, although higher antibody titers may be required for protection. Antibody responses to drifted A(H3N2) viruses following vaccination were influenced by multiple factors, including vaccine type and preexisting immunity from prior exposure., (Copyright © 2016 Levine et al.)

Whole inactivated virus (WIV) vaccines are widely used in the swine industry to reduce clinical disease against homologous influenza A virus (IAV) infection. In pigs experimentally challenged with antigenically distinct heterologous IAV of the same hemagglutinin subtype, WIV vaccinates have been shown to develop vaccine-associated enhanced respiratory disease (VAERD). We evaluated the impact of vaccine valency, age at vaccination, and duration between vaccination and challenge on the development of VAERD using vaccine containing δ1-H1N2 and challenge with pandemic H1N1 (pH1N1) virus. Pigs were vaccinated with monovalent WIV MN08 (δ1-H1N2) and bivalent (δ1-H1N2-H3N2 or δ1-H1N2-pH1N1) vaccines and then were challenged with pH1N1 at 3 weeks postboost (wpb). Another group was vaccinated with the same monovalent WIV and challenged at 6 wpb to determine if the time postvaccination plays a role in the development of VAERD. In a follow-up study, the impact of age of first WIV vaccination (at 4 versus 9 weeks of age) with a boost 3 weeks later (at 7 versus 12 weeks of age) was evaluated. A monovalent live-attenuated influenza virus (LAIV) vaccine administered at 4 and 7 weeks of age was also included. All mismatched WIV groups had significantly higher lung lesions than the LAIV, bivalent MN08-CA09, and control groups. Age of first vaccination or length of time between booster dose and subsequent challenge did not alter the development of VAERD in WIV-vaccinated pigs. Importantly, the mismatched component of the bivalent MN08-CA09 WIV did not override the protective effect of the matched vaccine component., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Respiratory syncytial virus (RSV) is the leading cause of severe respiratory disease in infants and children and represents an important global health burden for the elderly and the immunocompromised. Despite decades of research efforts, no licensed vaccine for RSV is available. We have developed virus-like particle (VLP)-based RSV vaccines assembled with the human metapneumovirus (hMPV) matrix protein (M) as the structural scaffold and the RSV fusion glycoprotein (F) in either the postfusion or prefusion conformation as its prime surface immunogen. Vaccines were composed of postfusion F, prefusion F, or a combination of the two conformations and formulated with a squalene-based oil emulsion as adjuvant. Immunization with these VLP vaccines afforded full protection against RSV infection and prevented detectable viral replication in the mouse lung after challenge. Analyses of lung cytokines and chemokines showed that VLP vaccination mostly induced the production of gamma interferon (IFN-γ), a marker of the Th1-mediated immune response, which is predominantly required for viral protection. Conversely, immunization with a formalin-inactivated RSV (FI-RSV) vaccine induced high levels of inflammatory chemokines and cytokines of the Th2- and Th17-mediated types of immune responses, as well as severe lung inflammation and histopathology. The VLP vaccines showed restricted production of these immune mediators and did not induce severe bronchiolitis or perivascular infiltration as seen with the FI-RSV vaccine. Remarkably, analysis of the serum from immunized mice showed that the VLP vaccine formulated using a combination of postfusion and prefusion F elicited the highest level of neutralizing antibody and enhanced the Th1-mediated immune response., (Copyright © 2016 Cimica et al.)

Shigella causes high morbidity and mortality worldwide, but there is no licensed vaccine for shigellosis yet. We evaluated the safety and immunogenicity of a formalin-inactivated whole-cell Shigella flexneri2a vaccine, Sf2aWC, given orally to adult volunteers. In a double-blind, placebo-controlled trial, 82 subjects were randomized to receive three doses of vaccine in dose escalation (2.6 ± 0.8 × 10(8), × 10(9), × 10(10), and × 10(11)vaccine particles/ml). Vaccine safety was actively monitored, and antigen-specific systemic and mucosal immune responses were determined in serum, antibody in lymphocyte supernatant (ALS), and fecal samples. Cytokines were measured in the serum. Sf2aWC was well tolerated and generally safe at all four dose levels. The vaccine resulted in a dose-dependent immune response. At the highest dose, the vaccine induced robust responses to lipopolysaccharide (LPS) in both serum and ALS samples. The highest magnitude and frequency of responses occurred after the first dose in almost all samples but was delayed for IgG in serum. Fifty percent of the vaccinees had a >4-fold increase in anti-LPS fecal antibody titers. Responses to invasion plasmid antigens (Ipa) were low. The levels of interleukin-17 (IL-17), IL-2, gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-10 were increased, and IL-8 was decreased immediately after first dose, but these changes were very transient. This phase I trial demonstrated that the Sf2aWC vaccine, a relatively simple vaccine concept, was safe and immunogenic. The vaccine elicited immune responses which were comparable to those induced by a live, attenuated Shigella vaccine that was protective in prior human challenge studies., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Although a number of studies have investigated and quantified immune correlates of protection against influenza in adults and children, data on immune protection in the elderly are sparse. A recent vaccine efficacy trial comparing standard-dose with high-dose inactivated influenza vaccine in persons 65 years of age and older provided the opportunity to examine the relationship between values of three immunologic assays and protection against community-acquired A/H3N2 influenza illness. The high-dose vaccine induced significantly higher antibody titers than the standard-dose vaccine for all assays. For the hemagglutination inhibition assay, a titer of 40 was found to correspond with 50% protection when the assay virus was antigenically well matched to the circulating virus--the same titer as is generally recognized for 50% protection in younger adults. A dramatically higher titer was required for 50% protection when the assay virus was a poor match to the circulating virus. With the well-matched virus, some protection was seen at the lowest titers; with the poorly matched virus, high levels of protection were not achieved even at the highest titers. Strong associations were also seen between virus neutralization test titers and protection, but reliable estimates for 50% protection were not obtained. An association was seen between titers of an enzyme-linked lectin assay for antineuraminidase N2 antibodies and protection; in particular, the proportion of treatment effect explained by assay titer in models that included both this assay and one of the other assays was consistently higher than in models that included either assay alone. (This study has been registered at ClinicalTrials.gov under registration no. NCT01427309.)., (Copyright © 2016 Dunning et al.)

Although experimental data regarding cross-protection of horse West Nile virus (WNV) vaccines against lineage 2 infections exist, the cross-protective efficacy of these vaccines under field conditions has not been demonstrated. This study was conducted to evaluate the capability of an inactivated lineage 1 vaccine (Equip WNV) to protect against natural infections from the Nea Santa-Greece-2010 lineage 2 strain. In total, 185 WNV-seronegative horses in Thessaloniki, Greece, were selected during 2 consecutive years (2011 and 2012); 140 were immunized, and 45 were used as controls. Horses were examined for signs compatible with WNV infection. Neutralizing antibody titers against the Greek strain and the PaAn001/France lineage 1 strain were determined in immunized horses. WNV circulation was detected during both years in the study area. It was estimated that 37% and 27% of the horses were infected during 2011 and 2012, respectively. Three control animals developed clinical signs, and the WNV diagnosis was confirmed. Signs related to WNV infection were not observed in the vaccinated animals. The nonvaccinated animals had a 7.58% ± 1.82% higher chance of exhibiting signs than immunized animals (P < 0.05). Neutralizing antibodies raised against both strains in all immunized horses were detectable 1 month after the initial vaccination course. The cross-protective capacity of the lowest titer (1:40) was evident in 19 animals which were subsequently infected and did not exhibit signs. Neutralizing antibodies were detectable until the annual booster, when strong anamnestic responses were observed (geometrical mean titer ratio [GMTR] for lineage 1 of 30.2; GMTR for lineage 2 of 27.5). The results indicate that Equip WNV is capable of inducing cross-protection against natural infections from a virulent lineage 2 WNV strain in horses., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

A pneumococcal whole-cell vaccine (WCV) confers T(H)17-mediated immunogenicity and reduces nasopharyngeal (NP) carriage in mice. Activation of Toll-like receptor 2 (TLR2) has been shown to be important for generating T(H)17 responses, and several lipidated pneumococcal proteins have TLR2-activating properties. Here we investigated the roles of TLR2 and lipidation of proteins in WCV-induced interleukin-17A (IL-17A) responses and protection against NP carriage. Immunization of Tlr2(-/-) mice with WCV conferred significantly lower IL-17A levels and reduced protection against NP carriage, compared to wild-type (WT) mice, suggesting that host TLR2 engagement is required for effective immunity and protection elicited by WCV immunization. Using a WCV with deletion of lgt, the gene encoding the enzyme required for lipidation and membrane attachment of prolipoproteins, we show that lipidation and membrane localization of these proteins are critical for the immunogenicity and protective efficacy of the WCV. To evaluate the roles of diacylglyceryl transferase (Lgt)-mediated processes in the recall of WCV-induced protective responses, we colonized WCV-immunized animals with a strain in which lgt was deleted. WCV-immunized animals still had significantly reduced colonization burdens, compared to control animals, which suggests that lipidation and membrane localization of pneumococcal prolipoproteins are less critical for the recall of the immune responses elicited by WCV immunization than for the priming of such responses. Elucidation of underlying immune mechanisms and the optimal characteristics of WCV formulations can help guide vaccine development and enhance our understanding of host-pneumococcus interactions., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Four vaccines for feline leukemia virus (FeLV) are available in the United States. This study's purpose was to compare the efficacy of Nobivac feline 2-FeLV (an inactivated, adjuvanted whole-virus vaccine) and PureVax recombinant FeLV (a live, canarypox virus-vectored vaccine) following FeLV challenge. Cats were vaccinated at 9 and 12 weeks with Nobivac feline 2-FeLV (group A, n = 11) or PureVax recombinant FeLV (group B, n = 10). Group C (n = 11) comprised unvaccinated controls. At 3 months postvaccination, cats were immunosuppressed and challenged with FeLV-A/61E. The outcomes measured were persistent antigenemia at 12 weeks postchallenge (PC) and proviral DNA and viral RNA at 3 to 9 weeks PC. Persistent antigenemia was observed in 0 of 11 cats in group A, 5 of 10 cats in group B, and 10 of 11 cats in group C. Group A was significantly protected compared to those in groups B (P < 0.013) and C (P < 0.0001). No difference was found between groups B and C (P > 0.063). The preventable fraction was 100% for group A and 45% for group B. At 9 weeks PC, proviral DNA and viral RNA were detected 1 of 11 cats in group A, 6 of 10 cats in group B, and 9 of 11 cats in group C. Nucleic acid loads were significantly lower in group A than in group C (P < 0.01). Group A had significantly lower proviral DNA loads than group B at weeks 6 to 9 (P < 0.02). The viral RNA loads were significantly lower in group A than in group B at weeks 7 to 9 (P < 0.01). The results demonstrate that Nobivac feline 2-FeLV-vaccinated cats were fully protected against persistent antigenemia and had significantly smaller amounts of proviral DNA and plasma viral RNA loads than PureVax recombinant FeLV-vaccinated cats and unvaccinated controls., (Copyright © 2015, Patel et al.)

The threat of varicella and herpes zoster in immunocompromised individuals necessitates the development of a safe and effective varicella-zoster virus (VZV) vaccine. The immune responses of guinea pigs to the intradermal (i.d.) or subcutaneous (s.c.) administration of a heat-inactivated or live VZV vaccine were investigated. Relative to nonimmunized animals, a single 399-PFU dose of vaccine induced nonsignificant increases in gamma interferon (IFN-γ), granzyme B, and perforin mRNA expression in the splenocytes of all groups, while two i.d. administrations of the inactivated vaccine increased IFN-γ mRNA expression significantly (P < 0.005). A single 1,995-PFU dose significantly increased the expression of IFN-γ mRNA in the groups receiving the vaccine either i.d. (P < 0.005) or s.c. (P < 0.05), that of granzyme B mRNA in the groups immunized i.d. with the inactivated (P < 0.005) or live (P < 0.005) vaccine, and that of perforin mRNA in the animals that received the inactivated vaccine i.d. (P < 0.005). Importantly, increases in the expression of IFN-γ (P = 0.025), granzyme B (P = 0.004), and perforin (P > 0.05) mRNAs were observed in the animals immunized i.d. with 1,995 PFU of inactivated vaccine relative to those immunized s.c. with the same dose. The proportion of animals expressing IFN-γ mRNA mirrored the proportion expressing IFN-γ protein (correlation coefficient of 0.88). VZV glycoprotein-specific and virus-neutralizing antibodies were produced with no significant intergroup differences. A booster i.d. administration of the 399-PFU dose of heat-inactivated vaccine enhanced the antibody responses. These results demonstrate that i.d. administration of an inactivated VZV vaccine can be an efficient mode of immunization against VZV., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Influenza whole inactivated virus (WIV) is more immunogenic and induces protective antibody responses compared with other formulations, like split virus or subunit vaccines, after intranasal mucosal delivery. Polyethyleneimine (PEI), an organic polycation, is widely used as a reagent for gene transfection and DNA vaccine delivery. Although PEI recently has demonstrated potent mucosal adjuvant activity for viral subunit glycoprotein antigens, its immune activity with H9N2 WIV is not well demonstrated. Here, mice were immunized intranasally with H9N2 WIV combined with PEI, and the levels of local respiratory tract and systemic immune responses were measured. Compared to H9N2 WIV alone, antigen-specific IgA levels in the local nasal cavity, trachea, and lung, as well as levels of IgG and its subtypes (IgG1 and IgG2a) in the serum, were strongly enhanced with the combination. Similarly, the activation and proliferation of splenocytes were markedly increased. In addition, PEI is superior as an H9N2 WIV delivery system due to its ability to greatly increase the viral adhesion to mucosal epithelial cells and to enhance the cellular uptake and endosomal escape of antigens in dendritic cells (DCs) and further significantly activate DCs to mature. Taken together, these results provided more insights that PEI has potential as an adjuvant for H9N2 particle antigen intranasal vaccination., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Ross River virus (RRV) is endemic in Australia and several South Pacific Islands. More than 90,000 cases of RRV disease, which is characterized by debilitating polyarthritis, were reported in Australia in the last 20 years. There is no vaccine available to prevent RRV disease. A phase 3 study was undertaken at 17 sites in Australia to investigate the safety and immunogenicity of an inactivated whole-virus Vero cell culture-derived RRV vaccine in 1,755 healthy younger adults aged 16 to 59 years and 209 healthy older adults aged ≥60 years. Participants received a 2.5-μg dose of Al(OH)(3)-adjuvanted RRV vaccine, with a second and third dose after 3 weeks and 6 months, respectively. Vaccine-induced RRV-specific neutralizing and total IgG antibody titers were measured after each immunization. Vaccine safety was monitored over the entire study period. The vaccine was safe and well-tolerated after each vaccination. No cases of arthritis resembling RRV disease were reported. The most frequently reported systemic reactions were headache, fatigue, and malaise; the most frequently reported injection site reactions were tenderness and pain. After the third immunization, 91.5% of the younger age group and 76.0% of the older age group achieved neutralizing antibody titers of ≥1:10; 89.1% of the younger age group and 70.9% of the older age group achieved enzyme-linked immunosorbent assay (ELISA) titers of ≥11 PanBio units. A whole-virus Vero cell culture-derived RRV vaccine is well tolerated in an adult population and induces antibody titers associated with protection from RRV disease in the majority of individuals. (This study is registered at www.clinicaltrials.gov under registration no. NCT01242670.)., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Interleukin-18 (IL-18) is an important cytokine involved in innate and acquired immunity. In this study, we cloned the full-length chicken IL-18 (ChIL-18) gene from specific-pathogen-free (SPF) chicken embryo spleen cells and provided evidence that the ChIL-18 gene in a recombinant plasmid was successfully expressed in chicken DT40 cells. ChIL-18 significantly enhanced gamma interferon (IFN-γ) mRNA expression in chicken splenocytes, which increased IFN-γ-induced nitric oxide (NO) synthesis by macrophages. The potential genetic adjuvant activity of the ChIL-18 plasmid was examined in chickens by coinjecting ChIL-18 plasmid and inactivated Newcastle disease virus (NDV) vaccine. ChIL-18 markedly elevated serum hemagglutination inhibition (HI) titers and anti-hemagglutinin-neuraminidase (anti-HN)-specific antibody levels, induced the secretion of both Th1- (IFN-γ) and Th2- (interleukin-4) type cytokines, promoted the proliferation of T and B lymphocytes, and increased the populations of CD3(+) T cells and their subsets, CD3(+) CD4(+) and CD3(+) CD8(+) T cells. Furthermore, a virus challenge revealed that ChIL-18 contributed to protection against Newcastle disease virus challenge. Taken together, our data indicate that the coadministration of ChIL-18 plasmid and NDV vaccine induces a strong immune response at both the humoral and cellular levels and that ChIL-18 is a novel immunoadjuvant suitable for NDV vaccination., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Detection of immunoglobulin M (IgM) antibodies has long been used as an important diagnostic tool for identifying active viral infections, but their relevance in later stages has not been clearly defined in vivo. In this study, we followed the kinetics, longevity, and function of influenza virus-specific IgM antibodies for 2 years following sublethal infection of mice with live mouse-adapted A/PR/8/34 virus or immunization with formalin-inactivated virus. These groups mounted robust protective immune responses and survived lethal challenges with 50 × 50% lethal dose (LD50) mouse-adapted A/PR/8/34 virus 600 days after the primary exposure. Surprisingly, the virus-specific IgM antibodies persisted along with IgG antibodies, and we found a significantly higher number of IgM-positive (IgM(+)) virus-specific plasma cells than IgG(+) plasma cells that persisted for at least 9 months postexposure. The IgM antibodies were functional as they neutralized influenza virus in the presence of complement just as well as IgG antibodies did., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Two cholera vaccines, sold as Shanchol and Dukoral, are currently available. This review presents a critical analysis of the protective efficacies of these vaccines. Children under 5 years of age are very vulnerable to cholera and account for the highest incidence of cholera cases and more than half of the resulting deaths. Both Shanchol and Dukoral are two-spaced-dose oral vaccines comprising large numbers of killed cholera bacteria. The former contains Vibrio cholerae O1 and O139 cells, and the latter contains V. cholerae O1 cells with the recombinant B subunit of cholera toxin. In a field trial in Kolkata (India), Shanchol, the preferred vaccine, protected 45% of the test subjects in all of the age groups and only 17% of the children under 5 years of age during the first year of surveillance. In a field trial in Peru, two spaced doses of Dukoral offered negative protection in children under 5 years of age and little protection (15%) in vaccinees over 6 years of age during the first year of surveillance. Little is known about Dukoral's long-term protective efficacy. Both of these vaccines have questionable compositions, using V. cholerae O1 strains isolated in 1947 that have been inactivated by heat and formalin treatments that may denature protein. Immunological studies revealed Dukoral's reduced and short-lived efficacy, as measured by several immunological endpoints. Various factors, such as the necessity for multiple doses, poor protection of children under 5 years of age, the requirement of a cold supply chain, production costs, and complex logistics of vaccine delivery, greatly reduce the suitability of either of these vaccines for endemic or epidemic cholera control in resource-poor settings., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Equine herpesvirus type 1 (EHV-1) is a major cause of winter pyrexia in racehorses in two training centers (Ritto and Miho) in Japan. Until the epizootic period of 2008-2009, a vaccination program using a killed EHV-1 vaccine targeted only susceptible 3-year-old horses with low antibody levels to EHV-1 antigens. However, because the protective effect was not satisfactory, in 2009-2010 the vaccination program was altered to target all 3-year-old horses. To evaluate the vaccine's efficacy, we investigated the number of horses with pyrexia due to EHV-1 or equine herpesvirus type 4 (EHV-4) infection or both and examined the vaccination coverage in the 3-year-old population and in the whole population before and after changes in the program. The mean (± standard deviation [SD]) estimated numbers of horses infected with EHV-1 or EHV-4 or both, among pyretic horses from 1999-2000 to 2008-2009 were 105 ± 47 at Ritto and 66 ± 44 at Miho. Although the estimated number of infected horses did not change greatly in the first period of the current program, it decreased from the second period, with means (±SD) of 21 ± 12 at Ritto and 14 ± 15 at Miho from 2010-2011 to 2012-2013. Vaccination coverage in the 3-year-old population was 99.4% at Ritto and 99.8% at Miho in the first period, and similar values were maintained thereafter. Coverage in the whole population increased more gradually than that in the 3-year-old population. The results suggest that EHV-1 epizootics can be suppressed by maintaining high vaccination coverage, not only in the 3-year-old population but also in the whole population., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Mycobacterium bovis causes animal tuberculosis (TB) in cattle, humans, and other mammalian species, including pigs. The goal of this study was to experimentally assess the responses of pigs with and without a history of tonsillectomy to oral vaccination with heat-inactivated M. bovis and challenge with a virulent M. bovis field strain, to compare pig and wild boar responses using the same vaccination model as previously used in the Eurasian wild boar (Sus scrofa), to evaluate the use of several enzyme-linked immunosorbent assays (ELISAs) and lateral flow tests for in vivo TB diagnosis in pigs, and to verify if these tests are influenced by oral vaccination with inactivated M. bovis. At necropsy, the lesion and culture scores were 20% to 43% higher in the controls than those in the vaccinated pigs. Massive M. bovis growth from thoracic tissue samples was observed in 4 out of 9 controls but in none of the 10 vaccinated pigs. No effect of the presence or absence of tonsils was observed on these scores, suggesting that tonsils are not involved in the protective response to this vaccine in pigs. The serum antibody levels increased significantly only after challenge. At necropsy, the estimated sensitivities of the ELISAs and dual path platform (DPP) assays ranged from 89% to 94%. In the oral mucosa, no differences in gene expression were observed in the control group between the pigs with and without tonsils. In the vaccinated group, the mRNA levels for chemokine (C-C motif) receptor 7 (CCR7), interferon beta (IFN-β), and methylmalonyl coenzyme A mutase (MUT) were higher in pigs with tonsils. Complement component 3 mRNA levels in peripheral blood mononuclear cells (PBMC) increased with vaccination and decreased after M. bovis challenge. This information is relevant for pig production in regions that are endemic for M. bovis and for TB vaccine research., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Studies were undertaken to manufacture a multivalent Shigella inactivated whole-cell vaccine that is safe, effective, and inexpensive. By using several formalin concentrations, temperatures, and incubation periods, an optimized set of inactivation conditions was established for Shigella flexneri 2a, S. sonnei, and S. flexneri 3a to produce inactivated whole cells expressing a full repertoire of Ipa proteins and lipopolysaccharide (LPS). The inactivation conditions selected were treatment with 0.2% formalin (S. flexneri 2a and 3a) or 0.6% formalin (S. sonnei) for 48 h at 25°C. Vaccine formulations prepared under different inactivation conditions, in different doses (10E5, 10E7, and 10E9 cells), and with or without the inclusion of double-mutant heat-labile toxin (dmLT) were evaluated in mice. Two intranasal immunizations with ≥10E7 inactivated whole cells resulted in high levels of anti-Invaplex and moderate levels of LPS-specific IgG and IgA in serum and in lung and intestinal wash samples. Addition of dmLT to the vaccine formulations did not significantly enhance humoral immunogenicity. Minimal humoral responses for IpaB, IpaC, or IpaD were detected after immunization with inactivated whole Shigella cells regardless of the vaccine inactivation conditions. In guinea pigs, monovalent formulations of S. flexneri 2a of 3a or S. sonnei consisting of 10E8, 10E9, or 10E10 cells were protective in a keratoconjunctivitis assay. A trivalent formulation provided protection against all three serotypes (S. flexneri 2a, P = 0.018; S. flexneri 3a, P = 0.04; S. sonnei, P < 0.0001). The inactivated Shigella whole-cell vaccine approach incorporates an uncomplicated manufacturing process that is compatible with multivalency and the future development of a broadly protective Shigella vaccine.

The immunogenicity and safety of an inactivated cell culture Japanese encephalitis vaccine (CC-JEV) were compared with those of an inactivated mouse brain-derived Japanese encephalitis vaccine (MB-JEV) in phase III clinical multicenter trials conducted in children. The vaccines contain the same Japanese encephalitis virus strain, the Beijing-1 strain. Two independent clinical trials (trials 1 and 2) were conducted. Trial 1 was conducted in 468 healthy children. Each subject was injected with 17 μg per dose of either CC-JEV or MB-JEV, and the immunogenicity and safety of the vaccines were investigated. Trial 1 showed that CC-JEV was more immunogenic and reactive than MB-JEV at the same dose. Therefore, to adjust the immunogenicity of CC-JEV to that of MB-JEV, a vaccine that has had a good track record regarding its efficacy for a long time, trial 2 was conducted in 484 healthy children. To improve the stability, CC-JEV was converted from a liquid type to a freeze-dried type of vaccine. Each subject was injected subcutaneously with either 4 μg per dose of CC-JEV, 8 μg per dose of CC-JEV, or 17 μg per dose of MB-JEV twice, at an interval of 2 to 4 weeks, followed by an additional booster immunization 1 to 15 months after the primary immunization. Based on the results of trial 2, 4 μg per dose of the freeze-dried CC-JEV (under the label Encevac) was selected as a substitute for the MB-JEV. Encevac was approved and launched in 2011 and has since been in use as a 2nd-generation Japanese encephalitis vaccine in Japan. (These studies have been registered at the JapicCTI under registration no. JapicCTI-132063 and JapicCTI-080586 for trials 1 and 2, respectively).

Bordetella pertussis fimbriae (Fim2 and Fim3) are components of a five-component acellular pertussis vaccine (diphtheria-tetanus-acellular pertussis vaccine [DTaP5]), and antibody responses to fimbriae have been associated with protection. We analyzed the IgG responses to individual Fim2 and Fim3 in sera remaining from a Swedish placebo-controlled efficacy trial that compared a whole-cell vaccine (diphtheria-tetanus-whole-cell pertussis vaccine [DTwP]), a two-component acellular pertussis vaccine (DTaP2), and DTaP5. One month following three doses of the Fim-containing vaccines (DTwP or DTaP5), anti-Fim2 geometric mean IgG concentrations were higher than those for anti-Fim3, with a greater anti-Fim2/anti-Fim3 IgG ratio elicited by DTaP5. We also determined the responses in vaccinated children following an episode of pertussis. Those who received DTaP5 showed a large rise in anti-Fim2 IgG, reflecting the predominant Fim2 serotype at the time. In contrast, those who received DTwP showed an equal rise in anti-Fim2 and anti-Fim3 IgG concentrations, indicating that DTwP may provide a more efficient priming effect for a Fim3 response following contact with B. pertussis. Anti-Fim2 and anti-Fim3 IgG concentrations were also determined in samples from two seroprevalence studies conducted in Sweden in 1997, when no pertussis vaccine was used and Fim2 isolates predominated, and in 2007, when either DTaP2 or DTaP3 without fimbriae was used and Fim3 isolates predominated. Very similar distributions of anti-Fim2 and anti-Fim3 IgG concentrations were obtained in 1997 and 2007, except that anti-Fim3 concentrations in 1997 were lower. This observation, together with the numbers of individuals with both anti-Fim2 and anti-Fim3 IgG concentrations, strongly suggests that B. pertussis expresses both Fim2 and Fim3 during infection.

Wildlife vaccination is increasingly being considered as an option for tuberculosis control. We combined data from laboratory trials and an ongoing field trial to assess the risk of an oral Mycobacterium bovis BCG vaccine and a prototype heat-inactivated Mycobacterium bovis preparation for Eurasian wild boar (Sus scrofa). We studied adverse reactions, BCG survival, BCG excretion, and bait uptake by nontarget species. No adverse reactions were observed after administration of BCG (n = 27) or inactivated M. bovis (n = 21). BCG was not found at necropsy (175 to 300 days postvaccination [n = 27]). No BCG excretion was detected in fecal samples (n = 162) or in urine or nasal, oral, or fecal swab samples at 258 days postvaccination (n = 29). In the field, we found no evidence of loss of BCG viability in baits collected after 36 h (temperature range, 11°C to 41°C). Camera trapping showed that wild boar (39%) and birds (56%) were the most frequent visitors to bait stations (selective feeders). Wild boar activity patterns were nocturnal, while diurnal activities were recorded for all bird species. We found large proportions of chewed capsules (29%) (likely ingestion of the vaccine) and lost baits (39%) (presumably consumed), and the proportion of chewed capsules showed a positive correlation with the presence of wild boar. Both results suggest proper bait consumption (68%). These results indicate that BCG vaccination in wild boar is safe and that, while bait consumption by other species is possible, this can be minimized by using selective cages and strict timing of bait deployment.

The determination of lot-to-lot consistency in the manufacturing process is a mandatory step in the clinical development of the novel enterovirus 71 (EV71) vaccine. A phase III, randomized, placebo-controlled, double-blind trial assessed the lot consistency, immunogenicity, and safety of the EV71 vaccine in children aged 6 to 59 months. Healthy children (n = 1,400) received one of three lots of the EV71 vaccine containing 400 U of EV71 antigen or a placebo at days 0 and 28. Blood samples were collected before dose 1 and at 28 days after dose 2 (day 56) for an anti-EV71 neutralizing antibody (NTAb) assay. The geometric mean titer (GMT) and the seropositivity rates (with titers of ≥1:8) were compared at day 56. After each dose, the solicited injection site and general adverse events (AEs) were recorded for 7 days, and unsolicited AEs were recorded for 28 days. At day 56, the seropositivity rates ranged from 99.7% to 100% for the vaccine groups. The NTAb GMTs for the vaccine were 140.3 (95% confidence interval [CI], 117.8 to 167.1), 141.5 (95% CI, 118.0 to 169.6), and 146.6 (95% CI, 122.5 to 175.3). The two-sided 95% CI of the log difference in GMTs between the pairs of lots were between -0.176 and 0.176, therefore meeting the predefined equivalence criteria. The percentages of subjects reporting any injection site AEs, general AEs, or serious AEs were similar across the four vaccination groups. In conclusion, the demonstration of consistency between the manufacturing lots confirms for the purposes of clinical development the reliability of the EV71 vaccine production process. (This study has been registered at ClinicalTrials.gov under registration no. NCT01636245.).

Antibody avidity for antigens following disease or vaccination increases with affinity maturation and somatic hypermutation. In this study, we followed children and adults in Bangladesh for 1 year following oral cholera vaccination and measured the avidity of antibodies to the T cell-dependent antigen cholera toxin B subunit (CTB) and the T cell-independent antigen lipopolysaccharide (LPS) in comparison with responses in other immunological measurements. Children produced CTB-specific IgG and IgA antibodies of high avidity following vaccination, which persisted for several months; the magnitudes of responses were comparable to those seen in adult vaccinees. The avidity of LPS-specific IgG and IgA antibodies in vaccinees increased significantly shortly after the second dose of vaccine but waned rapidly to baseline levels thereafter. CTB-specific memory B cells were present for only a short time following vaccination, and we did not find significant memory B cell responses to LPS in any age group. For older children, there was a significant correlation between CTB-specific memory T cell responses after the second dose of vaccine and CTB-specific IgG antibody avidity indices over the subsequent year. These findings suggest that vaccination induces a longer-lasting increase in the avidity of antibodies to a T cell-dependent antigen than is measured by a memory B cell response to that antigen and that early memory T cell responses correlate well with the subsequent development of higher-avidity antibodies.

This study investigated long-term cellular and humoral immunity against pertussis after booster vaccination of 4-year-old children who had been vaccinated at 2, 3, 4, and 11 months of age with either whole-cell pertussis (wP) or acellular pertussis (aP) vaccine. Immune responses were evaluated until 2 years after the preschool booster aP vaccination. In a cross-sectional study (registered trial no. ISRCTN65428640), blood samples were taken from wP- and aP-primed children prebooster and 1 month and 2 years postbooster. Pertussis vaccine antigen-specific IgG levels, antibody avidities, and IgG subclasses, as well as T-cell cytokine levels, were measured by fluorescent bead-based multiplex immunoassays. The numbers of pertussis-specific memory B cells and gamma interferon (IFN-γ)-producing T cells were quantified by enzyme-linked immunosorbent spot assays. Even 2 years after booster vaccination, memory B cells were still present and higher levels of pertussis-specific antibodies than prebooster were found in aP-primed children and, to a lesser degree, also in wP-primed children. The antibodies consisted mainly of the IgG1 subclass but also showed an increased IgG4 portion, primarily in the aP-primed children. The antibody avidity indices for pertussis toxin and pertactin in aP-primed children were already high prebooster and remained stable at 2 years, whereas those in wP-primed children increased. All measured prebooster T-cell responses in aP-primed children were already high and remained at similar levels or even decreased during the 2 years after booster vaccination, whereas those in wP-primed children increased. Since the Dutch wP vaccine has been replaced by aP vaccines, the induction of B-cell and T-cell memory immune responses has been enhanced, but antibody levels still wane after five aP vaccinations. Based on these long-term immune responses, the Dutch pertussis vaccination schedule can be optimized, and we discuss here several options.

Intraperitoneal vaccines using whole viable spores of the microsporidian Glugea anomala or Glugea hertwigi reduced the numbers of branchial xenomas by 80% and 91%, respectively, after a standard experimental infection of juvenile rainbow trout with the microsporidian Loma salmonae. Similar significant results were obtained when killed-spore preparations were used.

In this study, we assessed in humans the immunogenicity and safety of one dose (7.5 or 15 μg of hemagglutinin [HA]) of a whole-virion inactivated prepandemic influenza vaccine adjuvanted with aluminum hydroxide. The vaccine strain was made by reverse genetics from the highly pathogenic avian A/Chicken/Astana/6/05 (H5N1) clade 2.2 strain isolated from a dead bird in Kazakhstan. The humoral immune response was evaluated after a single vaccination by hemagglutination inhibition (HI) and microneutralization (MN) assays. The vaccine was safe and immunogenic, inducing seroconversion in 55% of the evaluated patients, with a geometric mean titer (GMT) of 17.1 and a geometric mean increase (GMI) of 3.42 after a dose of 7.5 μg in the HI test against the vaccine strain. The rate of seroconversion increased up to 70% when the dose of 15 μg was used. The percentages of individuals achieving anti-HA titers of ≥1:40 were 52.5% and 57.5% for the 7.5- and 15-μg dose groups, respectively. Similar results were obtained when antibodies were analyzed in an MN test. Substantial cross-neutralization titers (seroconversion in 35% and 52.5% of subjects in the two dose groups, respectively) were detected against heterologous clade 1 strain NIBRG14 (H5N1). Thus, one dose of this whole-virion prepandemic vaccine adjuvanted with aluminum has the potential to be effective against H5N1 viruses of different clades.

It is currently impossible to predict the next pandemic influenza virus strain. We have thus established a library of influenza viruses of all hemagglutinin and neuraminidase subtypes and their genes. In this article, we examine the applicability of a rapid production model for the preparation of vaccines against emerging pandemic influenza viruses. This procedure utilizes the influenza virus library, cell culture-based vaccine production, and intranasal administration to induce a cross-protective immune response. First, an influenza virus reassortant from the library, A/duck/Hokkaido/Vac-3/2007 (H5N1), was passaged 22 times (P22) in Madin-Darby canine kidney (MDCK) cells. The P22 virus had a titer of >2 ×10(8) PFU/ml, which was 40 times that of the original strain, with 4 point mutations, which altered amino acids in the deduced protein sequences encoded by the PB2 and PA genes. We then produced a formalin-inactivated whole-virion vaccine from the MDCK cell-cultured A/duck/Hokkaido/Vac-3/2007 (H5N1) P22 virus. Intranasal immunization of mice with this vaccine protected them against challenges with lethal influenza viruses of homologous and heterologous subtypes. We further demonstrated that intranasal immunization with the vaccine induced cross-reactive neutralizing antibody responses against the homotypic H5N1 influenza virus and its antigenic variants and cross-reactive cell-mediated immune responses to the homologous virus, its variants within a subtype, and even an influenza virus of a different subtype. These results indicate that a rapid model for emergency vaccine production may be effective for producing the next generation of pandemic influenza virus vaccines.

Influenza virus vaccination is recommended for children, but so far, active vaccination has not been achieved because most parents lack knowledge of vaccine safety and many doctors are reluctant to administer vaccine due to concerns that steroids might alter immunogenicity. The aim of this study was to compare the immunogenicity and safety of inactivated trivalent split influenza virus vaccine between children with recurrent wheezing and healthy children of the same age group. Sixty-eight healthy children and 62 children with recurrent wheezing took part in this study. Seroconversion rates, seroprotection rates, geometric mean titers (GMTs), and geometric mean titer ratios (GMTRs) were measured by a hemagglutination inhibition assay for the assessment of immunogenicity. Solicited and unsolicited local and systemic adverse events were measured for the assessment of safety. Regarding immunogenicity, the seroconversion and seroprotection rates showed no difference overall between healthy children and children with recurrent wheezing. Also, no difference was observed between steroid-treated and nontreated groups with recurrent wheezing. Generally, the GMTs after vaccination were higher in the one-dose vaccination groups for healthy children and children with recurrent wheezing, but the GMTRs revealed different results according to strain in the two groups. Regarding safety, solicited local and systemic adverse events showed no differences between healthy children and children with recurrent wheezing. This study demonstrates that inactivated split influenza virus vaccine is able to induce protective immune responses in healthy children, as observed in previous studies, as well as in children with recurrent wheezing who require frequent steroid treatment.

The 2009 pandemic H1N1 (pH1N1) influenza virus carried a swine-origin hemagglutinin (HA) that was closely related to the HAs of pre-1947 H1N1 viruses but highly divergent from the HAs of recently circulating H1N1 strains. Consequently, prior exposure to pH1N1-like viruses was mostly limited to individuals over the age of about 60 years. We related age and associated differences in immune history to the B cell response to an inactivated monovalent pH1N1 vaccine given intramuscularly to subjects in three age cohorts: 18 to 32 years, 60 to 69 years, and ≥70 years. The day 0 pH1N1-specific hemagglutination inhibition (HAI) and microneutralization (MN) titers were generally higher in the older cohorts, consistent with greater prevaccination exposure to pH1N1-like viruses. Most subjects in each cohort responded well to vaccination, with early formation of circulating virus-specific antibody (Ab)-secreting cells and ≥4-fold increases in HAI and MN titers. However, the response was strongest in the 18- to 32-year cohort. Circulating levels of HA stalk-reactive Abs were increased after vaccination, especially in the 18- to 32-year cohort, raising the possibility of elevated levels of cross-reactive neutralizing Abs. In the young cohort, an increase in MN activity against the seasonal influenza virus A/Brisbane/59/07 after vaccination was generally associated with an increase in the anti-Brisbane/59/07 HAI titer, suggesting an effect mediated primarily by HA head-reactive rather than stalk-reactive Abs. Our findings support recent proposals that immunization with a relatively novel HA favors the induction of Abs against conserved epitopes. They also emphasize the need to clarify how the level of circulating stalk-reactive Abs relates to resistance to influenza.

Commercial bacterins for Glässer's disease are widely used for the prevention of this disease caused by Haemophilus parasuis; however, the protective efficacy varies depending on the strain and serovar. Bacterial ghosts (BGs) are empty bacterial envelopes that, unlike classic bacterins, suffer no denaturing steps during their production. These properties may lead to superior protection. In this study, a BG vaccine generated from the Haemophilus parasuis serovar 5 reference strain Nagasaki was prepared and used to inoculate piglets. The efficacy of the BG vaccine was evaluated by clinical, bacteriological, serological, and postmortem examinations. Inactivated bacterin (IB) and a placebo control (PC) were compared with the BG vaccine in this study. The results showed that the piglets inoculated with the BG vaccine developed higher antibody activity and higher gamma interferon and interleukin 4 levels than those vaccinated with IB or those in the PC group after primary and secondary exposure to the antigens and challenge. CD4(+) T lymphocyte levels were observed to increase following secondary immunization more in the BG-vaccinated group than in the IB (P < 0.05) and PC (P < 0.05) groups. CD8(+) T lymphocyte levels increased dramatically in all three groups after challenge, and the differences between groups were all significant (P < 0.05). There were fewer tissue lesions and lower bacterial loads in the tissue homogenates in the BG group after challenge. The results suggest that higher CD4(+) T lymphocyte levels and both CD4(+) major histocompatibility complex class II-restricted Th1-type and Th2-type immune responses in the BG group are relevant for protection.

Current oral cholera vaccines induce lower levels of protective efficacy and shorter durations of protection in young children than in adults. Immunity against cholera is serogroup specific, and immune responses to Vibrio cholerae lipopolysaccharide (LPS), the antigen that mediates serogroup-specific responses, are associated with protection against disease. Despite this, responses against V. cholerae O-specific polysaccharide (OSP), a key component of the LPS responsible for specificity, have not been characterized in children. Here, we report a comparison of polysaccharide antibody responses in children from a region in Bangladesh where cholera is endemic, including infants (6 to 23 months, n = 15), young children (24 to 59 months, n = 14), and older children (5 to 15 years, n = 23) who received two doses of a killed oral cholera vaccine 14 days apart. We found that infants and young children receiving the vaccine did not mount an IgG, IgA, or IgM antibody response to V. cholerae OSP or LPS, whereas older children showed significant responses. In comparison to the vaccinees, young children with wild-type V. cholerae O1 Ogawa infection did mount significant antibody responses against OSP and LPS. We also demonstrated that OSP responses correlated with age in vaccinees, but not in cholera patients, reflecting the ability of even young children with wild-type cholera to develop OSP responses. These differences might contribute to the lower efficacy of protection rendered by vaccination than by wild-type disease in young children and suggest that efforts to improve lipopolysaccharide-specific responses might be critical for achieving optimal cholera vaccine efficacy in this younger age group.

Riemerella anatipestifer infections cause major economic losses in the duck industry. In this study, a trivalent inactivated vaccine of R. anatipestifer, including strains CH3 (serotype 1), NJ3 (serotype 2), and HXb2 (serotype 10), was developed. Animal experiments showed that the ducks that received two immunizations with the vaccine were 100% protected from challenge with strains from any of the three serotypes (1, 2, or 10). No death or clinical signs of diarrhea, tremors, or limb swelling were shown in the protected ducks. Also, no R. anatipestifer bacteria were isolated from the livers or brains of the protected ducks. Furthermore, no histopathological changes were observed in the liver, spleen, or brain samples from the protected ducks during histological examination. The ducks that received two immunizations with the vaccine generated high antibody titers of 1:3,200 to 1:6,400 against the three serotypes of strains. The vaccine significantly enhanced the production of gamma interferon (IFN-γ) and interleukin 2 (IL-2) after one immunization and enhanced the production of IL-4 and IL-10 after two immunizations. In addition, real-time PCR indicated that the expression of major histocompatibility complex I (MHC-I), as well as that of CD40 and CD154 molecules, was significantly increased after one immunization, and the expressions of both MHC-I and MHC-II molecules were increased after two immunizations. Our study indicates that the vaccine can induce both humoral and cellular immunities in ducks and offer effective protection against R. anatipestifer infection.

Enzootic pneumonia, resulting from infection with Mycoplasma hyopneumoniae, is of considerable economic importance to the pig industry and normally is controlled through active vaccination of piglets. We have demonstrated that administration of an inactivated Mycoplasma hyopneumoniae vaccine to piglets less than 1 week old is efficacious under field conditions and reduces the level of lung lesions observed in comparison to that in control pigs. Here, the results of two separate studies, one in piglets with and the second one in piglets without maternal antibodies, conducted to satisfy the requirements of the European Pharmacopoeia (monograph no. 07/2009:2448), are reported. Piglets received either minimal titer Suvaxyn MH-One or saline at less than 1 week of age and were challenged with Mycoplasma hyopneumoniae 2 weeks later. The number of lung lesions was recorded 4 weeks after challenge, and bronchial swab and lung tissue specimens were analyzed for quantification of Mycoplasma hyopneumoniae DNA. In the presence and absence of maternal antibodies, vaccination of piglets at less than 1 week of age was efficacious, with vaccinated piglets having significantly lower percentages of lung with lesions and lower Mycoplasma hyopneumoniae counts detected in bronchial swab and lung tissue specimens at necropsy. In conclusion, the vaccination of piglets at 1 week of age with Suvaxyn MH-One is efficacious in the presence of high levels of maternal antibodies.

Q fever is a zoonotic disease caused by infection with the bacterium Coxiella burnetii. Infection with C. burnetii results in humoral and cellular immune responses, both of which are thought to contribute to protection against subsequent infection. Whole-cell formalin-inactivated vaccines have also been shown to induce both humoral and cellular immunity and provide protection. Whether measurement of cellular or humoral immunity is a better indicator of immune protection is not known, and the duration of immunity induced by natural infection or vaccination is also poorly understood. To better understand the measurement and duration of C. burnetii immunity, 16 people vaccinated against Q fever (0.2 to 10.3 years before analysis) and 29 controls with a low risk of Q fever exposure were tested for immune responses to C. burnetii by an indirect fluorescent-antibody test (IFA) to measure circulating antibody and by a gamma interferon release assay (IGRA) to measure cellular immunity. Among vaccinated subjects, the IFA detected antibodies in 13/16, and the IGRA also detected positive responses in 13/16. All of the vaccinated subjects had a positive response in at least one of the assays, whereas 8/29 control subjects were positive in at least one assay. There was not a correlation between time since vaccination and responses in these assays. These results show that IFA and IGRA perform similarly in detection of C. burnetii immune responses and that Q fever vaccination establishes long-lived immune responses to C. burnetii.

Bacillus anthracis spores are the infectious form of the organism for humans and animals. However, the approved human vaccine in the United States is derived from a vegetative culture filtrate of a toxigenic, nonencapsulated B. anthracis strain that primarily contains protective antigen (PA). Immunization of mice with purified spore proteins and formalin-inactivated spores (FIS) from a nonencapsulated, nontoxigenic B. anthracis strain confers protection against B. anthracis challenge when PA is also administered. To investigate the capacity of the spore particle to act as a vaccine without PA, we immunized mice subcutaneously with FIS from nontoxigenic, nonencapsulated B. cereus strain G9241 pBCXO1(-)/pBC210(-) (dcG9241), dcG9241 ΔbclA, or 569-UM20 or with exosporium isolated from dcG9241. FIS vaccination provided significant protection of mice from intraperitoneal or intranasal challenge with spores of the virulent B. anthracis Ames or Ames ΔbclA strain. Immunization with dcG9241 ΔbclA FIS, which are devoid of the immunodominant spore protein BclA, provided greater protection from challenge with either Ames strain than did immunization with FIS from BclA-producing strains. In addition, we used prechallenge immune antisera to probe a panel of recombinant B. anthracis Sterne spore proteins to identify novel immunogenic vaccine candidates. The antisera were variably reactive with BclA and with 10 other proteins, four of which were previously tested as vaccine candidates. Overall our data show that immunization with FIS from nontoxigenic, nonencapsulated B. cereus strains provides moderate to high levels of protection of mice from B. anthracis Ames challenge and that neither PA nor BclA is required for this protection.

The immunogenicity of pandemic influenza A H1N1 virus (A/H1pdm) vaccine might be modified by prior seasonal trivalent influenza vaccine (sTIV) administration. We conducted a retrospective analysis of immunogenicity of 243 health care workers (number of sTIV-positive [sTIV(+)] subjects, 216; number of sTIV(-) subjects, 27) by hemagglutination inhibition. There was no significant difference in the ratios of antibody titers of ≥40 (41.2% versus 48.1%; P = 0.49) and fold increases in geometric mean titer (3.8 versus 4.5; P = 0.37). sTIV injected 7 to 10 days prior to A/H1pdm vaccine administration did not interfere with the immunogenicity of the latter.

Young children, older children, and adults develop comparable levels and durations of immunity following cholera. In comparison, young children receiving oral killed cholera vaccines (OCV) develop a lower level and shorter duration of protection than those of older children and adults. The reasons for this are unclear. We investigated OCV-induced memory T cell responses in younger and older children and compared responses to those in children with cholera. We found that patients with cholera developed significant levels of toxin-specific effector memory T cells (T(EM)) with follicular helper and gut-homing characteristics. Older children (6 to 14 years of age) receiving two doses of OCV containing recombinant cholera toxin B subunit (rCTB) had more modest T(EM) responses with follicular helper and gut-homing characteristics, but younger vaccinees (24 to 71 months of age) did not develop T(EM) responses. The T(EM) response correlated positively with subsequent IgG memory B cell responses specific to rCTB in older vaccinees. Cytokine analyses indicated that cholera patients developed significant Th1, Th17, and Th2 responses, while older children receiving vaccine developed more modest increases in Th1 and Th17 cells. Younger vaccinees had no increase in Th1 cells, a decrease in Th17 cells, and an increase in regulatory T (Treg) cells. Our findings suggest that T cell memory responses are markedly diminished in children receiving OCV, especially young children, compared to responses following naturally acquired cholera, and that these differences affect subsequent development of memory B cell responses. These findings may explain the lower efficacy and shorter duration of protection afforded by OCV in young children.

The antigenicity of seasonal human influenza virus changes continuously; thus, a cross-protective influenza vaccine design needs to be established. Intranasal immunization with an influenza split-virion (SV) vaccine and a mucosal adjuvant induces cross-protection; however, no mucosal adjuvant has been assessed clinically. Formalin-inactivated intact human and avian viruses alone (without adjuvant) induce cross-protection against the highly pathogenic H5N1 avian influenza virus. However, it is unknown whether seasonal human influenza formalin-inactivated whole-virion (WV) vaccine alone induces cross-protection against strains within a subtype or in a different subtype of human influenza virus. Furthermore, there are few reports comparing the cross-protective efficacy of the WV vaccine and SV vaccine-mucosal adjuvant mixtures. Here, we found that the intranasal human influenza WV vaccine alone induced both the innate immune response and acquired immune response, resulting in cross-protection against drift variants within a subtype of human influenza virus. The cross-protective efficacy conferred by the WV vaccine in intranasally immunized mice was almost the same as that conferred by a mixture of SV vaccine and adjuvants. The level of cross-protective efficacy was correlated with the cross-reactive neutralizing antibody titer in the nasal wash and bronchoalveolar fluids. However, neither the SV vaccine with adjuvant nor the WV vaccine induced cross-reactive virus-specific cytotoxic T-lymphocyte activity. These results suggest that the intranasal human WV vaccine injection alone is effective against variants within a virus subtype, mainly through a humoral immune response, and that the cross-protection elicited by the WV vaccine and the SV vaccine plus mucosal adjuvants is similar.

Porcine hemagglutinating encephalomyelitis (PHE) is caused by the coronavirus hemagglutinating encephalomyelitis virus (PHE-CoV), and the recent, rapid spread of PHE-CoV in piglets from many countries emphasizes the urgent need for a PHE-CoV vaccine. Here we use a murine model for evaluation of the induction of humoral and cellular immune responses by inactivated and PHE-CoV DNA vaccines in order to define the immune correlates for protection against PHE-CoV. The inactivated vaccine was composed of purified PHE-CoV and aluminum hydroxide gel (alum), which was chosen as an adjuvant because of its long history of safety for human use. The PHE-CoV DNA vaccine was constructed by subcloning the S1 gene of PHE-CoV into the pVAX1 vector to create the recombinant plasmid pV-S1. Our results showed that the inactivated PHE-CoV vaccine (IPV) elicited a high level of humoral immunity, resulting in good protection efficacy against PHE-CoV challenge. The IPV induced the IgG1 subclass of serum antibodies and expression of the cytokine interleukin-4 (IL-4), suggesting that the IPV generated a predominantly Th2-type immune response. The DNA vaccine was found to mediate primarily a cellular immune response with high levels of IgG2a and the cytokines IL-2 and gamma interferon (IFN-γ). However, mice that were vaccinated twice with the DNA vaccine and boosted with the IPV could mount a sufficient neutralizing antibody response against live PHE-CoV, with little variation in IgG1 and IgG2a levels, and showed high levels of IL-2 and IL-4. This response may activate both B and T cells to mount a specific humoral and cellular immune response that could, in turn, elicit a phagocyte-mediated defense against PHE-CoV infections to achieve viral clearance.

Current oral cholera vaccines induce lower protective efficacy and shorter duration of protection against cholera than wild-type infection provides, and this difference is most pronounced in young children. Despite this, there are limited data comparing immune responses in children following wild-type disease versus vaccination, especially with regard to memory responses associated with long-term immunity. Here, we report a comparison of immune responses in young children (2 to 5 years of age; n = 20) and older children (6 to 17 years of age; n = 20) given two doses of an oral killed cholera vaccine containing recombinant cholera toxin B subunit (CtxB) 14 days apart and compare these responses to those induced in similarly aged children recovering from infection with Vibrio cholerae O1 Ogawa in Bangladesh. We found that the two vaccine groups had comparable vibriocidal and lipopolysaccharide (LPS)-specific plasma antibody responses. Vaccinees developed lower levels of IgG memory B cell (MBC) responses against CtxB but no significant MBC responses against LPS. In contrast, children recovering from natural cholera infection developed prominent LPS IgG and IgA MBC responses, as well as CtxB IgG MBC responses. Plasma LPS IgG, IgA, and IgM responses, as well as vibriocidal responses, were also significantly higher in children following disease than after vaccination. Our findings suggest that acute and memory immune responses following oral cholera vaccination in children are significantly lower than those observed following wild-type disease, especially responses targeting LPS. These findings may explain, in part, the lower efficacy of oral cholera vaccination in children.

There are currently no effective vaccines for visceral leishmaniasis, the second most deadly parasitic infection in the world. Here, we describe a novel whole-cell vaccine approach using Leishmania infantum chagasi promastigotes treated with the psoralen compound amotosalen (S-59) and low doses of UV A radiation. This treatment generates permanent, covalent DNA cross-links within parasites and results in Leishmania organisms termed killed but metabolically active (KBMA). In this report, we characterize the in vitro growth characteristics of both KBMA L. major and KBMA L. infantum chagasi. Concentrations of S-59 that generate optimally attenuated parasites were identified. Like live L. infantum chagasi, KBMA L. infantum chagasi parasites were able to initially enter liver cells in vivo after intravenous infection. However, whereas live L. infantum chagasi infection leads to hepatosplenomegaly in mice after 6 months, KBMA L. infantum chagasi parasites were undetectable in the organs of mice at this time point. In vitro, KBMA L. infantum chagasi retained the ability to enter macrophages and induce nitric oxide production. These characteristics of KBMA L. infantum chagasi correlated with the ability to prophylactically protect mice via subcutaneous vaccination at levels similar to vaccination with live, virulent organisms. Splenocytes from mice vaccinated with either live L. infantum chagasi or KBMA L. infantum chagasi displayed similar cytokine patterns in vitro. These results suggest that KBMA technology is a potentially safe and effective novel vaccine strategy against the intracellular protozoan L. infantum chagasi. This approach may represent a new method for whole-cell vaccination against other complex intracellular pathogens.

With the recent pandemic of influenza A (H1N1) and vaccine shortages, there has been considerable interest in developing influenza vaccines with reduced doses, allowing for increased production capacity. Here we report a prospective, randomized, double-blind, single-center clinical trial of a reduced-dose whole-virion inactivated, adjuvanted influenza vaccine in adult and elderly volunteers. A total of 234 subjects, including 120 adults (18 to 60 years of age) and 114 elderly subjects (>60 years of age) were enrolled to receive either 6 μg or the conventional 15-μg dose of seasonal trivalent influenza vaccines. The subjects were followed for safety analysis, and serum samples were obtained to assess immunogenicity by hemagglutination inhibition testing. The subjects developed antibody responses against the seasonal influenza A virus H1N1 and H3N2 strains, as well as the seasonal influenza B virus included in the vaccines. Single doses of 6 μg fulfilled licensing criteria for seasonal influenza vaccines. No significant differences in rates of seroconversion or seroprotection or in geometric mean titers were found between the two dosage levels. All adverse events were rare, mild, and transient. We found that the present reduced-dose vaccine is safe and immunogenic in healthy adult and elderly subjects and triggers immune responses that comply with licensing criteria.

Inhalation of Yersinia pestis causes pneumonic plague, which rapidly progresses to death. A previously licensed killed whole-cell vaccine is presently unavailable due to its reactogenicity and inconclusive evidence of efficacy. The present study now shows that vaccination intranasally (i.n.) with inactivated Y. pestis CO92 (iYp) adjuvanted with interleukin-12 (IL-12) followed by an i.n. challenge with a lethal dose of Y. pestis CO92 prevented bacterial colonization and protected 100% of mice from pneumonic plague. Survival of the vaccinated mice correlated with levels of systemic and lung antibodies, reduced pulmonary pathology and proinflammatory cytokines, and the presence of lung lymphoid cell aggregates. Protection against pneumonic plague was partially dependent upon Fc receptors and could be transferred to naïve mice with immune mouse serum. On the other hand, protection was not dependent upon complement, and following vaccination, depletion of CD4 and/or CD8 T cells before challenge did not affect survival. In summary, the results demonstrate the safety, immunogenicity, and protective efficacy of i.n. administered iYp plus IL-12 in a mouse model of pneumonic plague.

Porcine circovirus type 2 (PCV2) is the obligate infectious agent in postweaning multisystemic wasting syndrome (PMWS) of pigs. To control PMWS, we vaccinated dams at 4 and 2 weeks before pregnancy and again in the 12th week of gestation with an inactivated PCV2 vaccine (Circovac). Two producer farms run under the control of Swiss Swine Health Organization were selected for the experiment. Previously, in one farm PMWS was diagnosed on pigs after weaning, whereas in the other farm, pigs wasted during the fattening period. For the experiments 113 dams were randomly vaccinated, and 111 dams were sham injected. Vaccination increased serum antibodies in dams 3- to 9-fold, accompanied by serum antibody titer increases in their offspring. In the sixth week of life, progeny from vaccinated dams had about the same IgG antibody titers as progeny of unvaccinated dams at the third day of life. In sera of vaccinated dams only low concentrations of PCV2 DNA were detected, and no progeny developed PMWS. Interestingly, at day 56 four progeny of unvaccinated dams tested positive for anti-PCV2 IgM antibodies, indicating a primary infection with PCV2. Of economic importance is the observation that progeny of vaccinated dams had a significantly higher daily weight gain in the fattening period (farm X, +51 g/day; farm Y, +30 g/day) and thus a shortened fattening period of about 6 days compared to progeny of controls. To our knowledge this is the first demonstration of subclinical circovirus infection and its effects on growth performance of fattening pigs by vaccination of dams.

The protective immunity of baculovirus displaying influenza virus hemagglutinin (BacHA) against influenza 2009 H1N1 virus infection in a murine model was investigated. The results showed that mice vaccinated with live BacHA or an inactive form of adjuvanted BacHA had enhanced specific antibody responses and induced protective immunity against 2009 H1N1 virus infection, suggesting the potential of baculovirus as a live or inactivated vaccine.

There is an urgent need for identification of a new adjuvant capable of selectively promoting an efficient immune response for use with vaccines and especially subunit vaccines. Our pervious study showed that Bursopentine (BP5) is a novel immunomodulatory peptide and has the ability to significantly stimulate an antigen-specific immune response in mice. In this study, the potential adjuvant activities of BP5 were examined in chickens by coinjection of BP5 and an inactivated avian influenza virus (AIV) (A/Duck/Jiangsu/NJ08/05 [AIV H9N2 subtype]). The results suggested that BP5 markedly elevated serum hemagglutination inhibition (HI) titers and antigen-specific antihemagglutinin (anti-HA) antibody (IgG) levels, induced both Th1 (interleukin 2 [IL-2] and gamma interferon [IFN-γ])- and Th2 (IL-4)-type cytokines, promoted the proliferation of peripheral blood lymphocytes, and increased populations of CD3(+) T cells and their subsets CD4(+) (CD3(+) CD4(+)) T cells and CD8(+) (CD3(+) CD8(+)) T cells. Furthermore, a virus challenge experiment revealed that BP5 contributes to protection against homologous avian influenza virus challenge by reducing viral replication in chicken lungs. This study indicates that the combination of inactivated AIVs and BP5 gives a strong immune response at both the humoral and cellular levels and implies that BP5 is a novel immunoadjuvant suitable for vaccine design.

In 2005, European Commission directive 2005/744/EC allowed controlled vaccination against avian influenza (AI) virus of valuable avian species housed in zoos. In 2006, 15 Spanish zoos and wildlife centers began a vaccination program with a commercial inactivated H5N9 vaccine. Between November 2007 and May 2008, birds from 10 of these centers were vaccinated again with a commercial inactivated H5N3 vaccine. During these campaigns, pre- and postvaccination samples from different bird orders were taken to study the response against AI virus H5 vaccines. Sera prior to vaccinations with both vaccines were examined for the presence of total antibodies against influenza A nucleoprotein (NP) by a commercial competitive enzyme-linked immunosorbent assay (cELISA). Humoral responses to vaccination were evaluated using a hemagglutination inhibition (HI) assay. In some taxonomic orders, both vaccines elicited comparatively high titers of HI antibodies against H5. Interestingly, some orders, such as Psittaciformes, which did not develop HI antibodies to either vaccine formulation when used alone, triggered notable HI antibody production, albeit in low HI titers, when primed with H5N9 and during subsequent boosting with the H5N3 vaccine. Vaccination with successive heterologous vaccines may represent the best alternative to widely protect valuable and/or endangered bird species against highly pathogenic AI virus infection.

The mediators of protective immunity against cholera are currently unknown, but memory B-cell responses may play a central role in facilitating long-term and anamnestic responses against Vibrio cholerae, the cause of cholera. We compared memory B-cell responses in adults with natural cholera in Bangladesh (n = 70) to responses in Bangladeshi adults after one-dose (n = 30) or two-dose (n = 30) administration of an oral killed cholera vaccine, WC-rBS (Dukoral; Crucell), assessing the responses at the acute stage of disease or prevaccination and then on days 3, 30, 90, 180, 270, and 360. Individuals with natural cholera developed prominent vibriocidal and plasma anti-cholera toxin B subunit (CtxB) and lipopolysaccharide (LPS) IgG and IgA responses, but these responses returned to baseline by 1 year of follow-up. Vaccinees developed plasma anti-CtxB and anti-LPS IgG and IgA responses that were generally comparable to those in individuals recovering from natural disease, but vibriocidal responses were lower in vaccinees than in infected patients. Individuals recovering from natural disease developed memory B-cell IgG and IgA anti-CtxB and anti-LPS responses by day 30, and these responses were detectable through at least days 180 to 360. In contrast, we detected no IgA or IgG memory B-cell responses to LPS in vaccinees; anti-CtxB IgA responses were only detectable on day 30, and anti-CtxB IgG responses were detectable until days 90 to 180, compared to days 270 to 360 in patients. These findings may explain in part the relatively short-term protection afforded by oral cholera vaccination compared to natural disease.

Chronic infection of cattle with Leptospira borgpetersenii serovar Hardjo reduces animal production through reproductive failure and presents a persistent health threat to workers in the animal industry. Cattle are maintenance hosts for serovar Hardjo, and development of vaccines that establish long-term protective immunity has been problematic; induction of high titers of anti-serovar Hardjo antibody does not appear to be protective. Rather, development of an antigen-specific Th1 response appears to be critical for limiting renal colonization and urinary shedding of bacteria. In this study we compared two monovalent killed bacterial cell vaccines to assess long-term (12 months) protection against live serovar Hardjo challenge. Although neither vaccine prevented infection, renal colonization and urinary shedding of bacteria were reduced compared to those of control animals. Increased proliferation of CD4(+), CD8(+), and γδ T cells from vaccinated, but not control, animals was detected. In addition, NK cells from vaccinated animals and from all animals following infection, when exposed to antigen ex vivo, demonstrated a gamma interferon (IFN-γ) recall response. We propose that programming NK cells to respond quickly to L. borgpetersenii serovar Hardjo infection may be an important step toward developing protective immunity.