Higher Serum IgG Titers Research Articles

Intranasal SARS-CoV-2 Omicron variant vaccines elicit humoral and cellular mucosal immunity in female mice

In order to prevent the emergence and spread of future variants of concern of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), developing vaccines capable of stopping transmission is crucial. The SARS-CoV-2 vaccine NDV-HXP-S can be administered live intranasally (IN) and thus induce protective immunity in the upper respiratory tract. The vaccine is based on Newcastle disease virus (NDV) expressing a stabilised SARS-CoV-2 spike protein. NDV-HXP-S can be produced as influenza virus vaccine at low cost in embryonated chicken eggs. The NDV-HXP-S vaccine was genetically engineered to match the Omicron variants of concern (VOC) BA.1 and BA.5 and tested as an IN two or three dose vaccination regimen in female mice. Furthermore, female mice intramuscularly (IM) vaccinated with mRNA-lipid nanoparticles (LNPs) were IN boosted with NDV-HXP-S. Systemic humoral immunity, memory T cell responses in the lungs and spleens as well as immunoglobulin A (IgA) responses in distinct mucosal tissues were characterised. NDV-HXP-S Omicron variant vaccines elicited high mucosal IgA and serum IgG titers against respective SARS-CoV-2 VOC in female mice following IN administration and protected against challenge from matched variants. Additionally, antigen-specific memory B cells and local T cell responses in the lungs were induced. Host immunity against the NDV vector did not interfere with boosting. Intramuscular vaccination with mRNA-LNPs was enhanced by IN NDV-HXP-S boosting resulting in improvement of serum neutralization titers and induction of mucosal immunity. We demonstrate that NDV-HXP-S Omicron variant vaccines utilised for primary immunizations or boosting efficiently elicit humoral and cellular immunity. The described induction of systemic and mucosal immunity has the potential to reduce infection and transmission. This work was partially funded by the NIAIDCenters of Excellence for Influenza Research and Response (CEIRR) and by the NIAID Collaborative Vaccine Innovation Centers and by institutional funding from the Icahn School of Medicine at Mount Sinai. See under Acknowledgements for details.

Photochemical inactivation as an alternative method to produce a whole-cell vaccine for uropathogenic Escherichia coli (UPEC).

Lower urinary tract infection (UTI), caused primarily by uropathogenic Escherichia coli, represents a significant health burden, accounting for 7 million primary care and 1 million emergency room visits annually in the United States. Women and the elderly are especially susceptible and recurrent infection (rUTI) is common in those populations. Lower UTI can lead to life-threatening systemic infection. UTI burden is manifested by healthcare dollars spent (1.5 billion annually), quality of life impact, and resistant strains emerging from antibiotic overuse. A safe and effective vaccine to prevent rUTI would address a substantial healthcare issue. Vaccines comprised of inactivated uropathogenic bacteria have yielded encouraging results in clinical trials but improvements that enhance vaccine performance are needed. To that end, we focused on inactivation methodology and provided data to support photochemical inactivation, which targets nucleic acid, as a promising alternative to conventional protein-damaging inactivation methods to improve whole-cell UTI vaccines.

A candidate glycoconjugate vaccine induces protective antibodies in the serum and intestinal secretions, antibody recall response and memory T cells and protects against both typhoidal and non-typhoidal Salmonella serovars.

Human Salmonella infections pose significant public health challenges globally, primarily due to low diagnostic yield of systemic infections, emerging and expanding antibiotic resistance of both the typhoidal and non-typhoidal Salmonella strains and the development of asymptomatic carrier state that functions as a reservoir of infection in the community. The limited long-term efficacy of the currently licensed typhoid vaccines, especially in smaller children and non-availability of vaccines against other Salmonella serovars necessitate active research towards developing a multivalent vaccine with wider coverage of protection against pathogenic Salmonella serovars. We had earlier reported immunogenicity and protective efficacy of a subunit vaccine containing a recombinant outer membrane protein (T2544) of Salmonella Typhi in a mouse model. This was achieved through the robust induction of serum IgG, mucosal secretory IgA and Salmonella-specific cytotoxic T cells as well as memory B and T cell response. Here, we report the development of a glycoconjugate vaccine, containing high molecular weight complexes of Salmonella Typhimurium O-specific polysaccharide (OSP) and recombinant T2544 that conferred simultaneous protection against S. Typhi, S. Paratyphi, S. Typhimurium and cross-protection against S. enteritidis in mice. Our findings corroborate with the published studies that suggested the potential of Salmonella OSP as a vaccine antigen. The role of serum antibodies in vaccine-mediated protection is suggested by rapid seroconversion with high titers of serum IgG and IgA, persistently elevated titers after primary immunization along with a strong antibody recall response with higher avidity serum IgG against both OSP and T2544 and significantly raised SBA titers of both primary and secondary antibodies against different Salmonella serovars. Elevated intestinal secretory IgA and bacterial motility inhibition by the secretory antibodies supported their role as well in vaccine-induced protection. Finally, robust induction of T effector memory response indicates long term efficacy of the candidate vaccine. The above findings coupled with protection of vaccinated animals against multiple clinical isolates confirm the suitability of OSP-rT2544 as a broad-spectrum candidate subunit vaccine against human infection due to typhoidal and non-typhoidal Salmonella serovars.

Intranasal Single-Replication Influenza Vector Induces Cross-Reactive Serum and Mucosal Antibodies against SARS-CoV-2 Variants.

Current SARS-CoV-2 vaccines provide protection for COVID-19-associated hospitalization and death, but remain inefficient at inhibiting initial infection and transmission. Despite updated booster formulations, breakthrough infections and reinfections from emerging SARS-CoV-2 variants are common. Intranasal vaccination to elicit mucosal immunity at the site of infection can improve the performance of respiratory virus vaccines. We developed SARS-CoV-2 M2SR, a dual SARS-CoV-2 and influenza vaccine candidate, employing our live intranasal M2-deficient single replication (M2SR) influenza vector expressing the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein of the prototype strain, first reported in January 2020. The intranasal vaccination of mice with this dual vaccine elicits both high serum IgG and mucosal IgA titers to RBD. Sera from inoculated mice show that vaccinated mice develop neutralizing SARS-CoV-2 antibody titers against the prototype and Delta virus strains, which are considered to be sufficient to protect against viral infection. Moreover, SARS-CoV-2 M2SR elicited cross-reactive serum and mucosal antibodies to the Omicron BA.4/BA.5 variant. The SARS-CoV-2 M2SR vaccine also maintained strong immune responses to influenza A with high titers of anti H3 serum IgG and hemagglutination inhibition (HAI) antibody titers corresponding to those seen from the control M2SR vector alone. With a proven safety record and robust immunological profile in humans that includes mucosal immunity, the M2SR influenza viral vector expressing key SARS-CoV-2 antigens could provide more efficient protection against influenza and SARS-CoV-2 variants.

Immunization with Tp0954, an adhesin of Treponema pallidum, provides protective efficacy in the rabbit model of experimental syphilis.

Syphilis, a chronic multisystemic disease caused by spirochete Treponema pallidum subspecies pallidum infection, continues to be a serious global health problem and congenital syphilis remains a major cause of adverse outcomes in pregnancy in developing countries. The development of an effective vaccine is the most cost-effective way to eliminate syphilis, but so far has been elusive. Here, we evaluated the immunogenicity and protective efficacy of Tp0954, a T. pallidum placental adhesin, as a potential vaccine candidate in a New Zealand White rabbit model of experimental syphilis. Animals immunized with recombinant Tp0954 (rTp0954) produced high titers of Tp0954-specific serum IgG, high levels of IFN-γ from splenocytes and specific splenocyte proliferation response when compared to control animals immunized with PBS and Freund's adjuvant (FA). Furthermore, rTp0954 immunization significantly delayed the development of cutaneous lesions, promoted inflammatory cellular infiltration at the primary lesion sites, as well as inhibited T. pallidum dissemination to distal tissues or organs when compared with that of the control animals. In addition, the naïve rabbits receiving popliteal lymph nodes from Tp0954-immunized, T. pallidum-challenged animals were not infected by T. pallidum, confirming sterile immunity. These findings suggest that Tp0954 is a potential vaccine candidate against syphilis.

263. Antibody Spill-Over vs. True Coccidioidal Meningitis in a Patient with HIV/AIDS and Disseminated Coccidioidomycosis

Abstract Background The diagnosis of coccidioidal meningitis merits life-long antifungal therapy given high rates of disease recurrence. Accurate diagnosis is important. Antibody spill-over into cerebrospinal fluid (CSF) can happen when serum titers are high. We present a case of antibody spill-over vs. true fluconazole-resistant coccidioidal meningitis. Methods A 49-year-old man presented with 6 months of intermittent fever, myalgias, decreased appetite, vomiting, diarrhea, unsteadiness and 60-pound weight loss. He was recently diagnosed with HIV and a prior lymph node biopsy had grown Coccidioides immitis (C. i) for which he was given fluconazole 100 mg twice daily. Figure 1. Timeline of Coccidioides immitis lab results in relation to treatment regimen. CF: Complement fixation, EIA: Enzyme immunoassay for Coccidioides antigen, CSF: Cerebrospinal fluid, c/mL: copies/mL, R: resistant, S: susceptible. Results Vitals revealed a temperature of 102°F. He was cachectic and a 0.5 cm right supraclavicular lymph node was palpable. No meningeal signs were appreciated. CD4 count was 50/µL (18%), HIV-1 viral load 2,969,945 copies/mL. Computed tomography (CT) of the abdomen/pelvis suggested lung and spleen involvement. Serum C. i enzyme immunoassay (EIA) was 1.38 ng/mL, immunodiffusion (ID) was positive and complement fixation (CF) titer was 1:256. C. i was isolated from expectorated sputum. CSF cell count was normal, but ID was positive and CF titer was 1:2 however, lab reported concern for spill-over due to high serum IgG titers. He left against medical advice with fluconazole 400 mg daily. He was hospitalized a month later for failure to thrive. MRI head revealed enlarged lateral and third ventricles with increased periventricular hyperintensity concerning for coccidioidal meningitis. Repeat serum studies were stable. CSF revealed CF 1:4 and C. i antigen by EIA 1.31ng/mL, distinguishing between spill-over and meningitis. Susceptibility results showed resistance to fluconazole and amphotericin B with minimum inhibitory concentrations (MICs) of 50 and 4 respectively, posaconazole susceptibility (MIC < 1) and itraconazole borderline (MIC 3.7). Despite amphotericin B resistance, it was used for bridge to posaconazole. ART was initiated after concern for immune reconstitution had resolved. Conclusion This case highlights the difficulty in making an accurate diagnosis of coccidioidal meningitis. It also describes a fluconazole-resistant C. i isolate in the setting of prolonged low-dose fluconazole therapy. Disclosures All Authors: No reported disclosures

Serum IgG titers against periodontal pathogens are associated with cerebral hemorrhage growth and 3-month outcome.

To assess the influence of periodontal disease on cerebral hemorrhage and its clinical course, we examined the association of the serum IgG titer of periodontal pathogens with hemorrhage growth and 3-month outcome. We consecutively enrolled 115 patients with acute cerebral hemorrhage (44 females, aged 71.3 ± 13.1 years) and used ELISA to evaluate the serum IgG titers of 9 periodontal pathogens: Porphyromonas gingivalis, Aggregatibacter (A.) actinomycetemcomitans, Prevotella intermedia, Prevotella nigrescens, Fusobacterium (F.) nucleatum, Treponema denticola, Tannerella forsythensis, Campylobacter rectus, and Eikenella corrodens. Significant hematoma growth was defined as an increase in the volume of >33% or an absolute increase in the volume of >12.5 mL. A poor outcome was defined as a 3 or higher on the modified Rankin Scale. We observed hemorrhage growth in 13 patients (11.3%). Multivariate analysis revealed that increased IgG titers of A. actinomycetemcomitans independently predicted the elevated hemorrhage growth (odds ratio 5.26, 95% confidence interval 1.52-18.25, p = 0.01). Notably, augmented IgG titers of F. nucleatum but not A. actinomycetemcomitans led to a poorer 3-month outcome (odds ratio 7.86, 95% confidence interval 1.08-57.08, p = 0.04). Thus, we demonstrate that elevated serum IgG titers of A. actinomycetemcomitans are an independent factor for predicting cerebral hemorrhage growth and that high serum IgG titers of F. nucleatum may predict a poor outcome in patients with this disease. Together, these novel data reveal how systemic periodontal pathogens may affect stroke patients, and, should, therefore, be taken into consideration in the management and treatment of these individuals.

Protection against a chlamydial respiratory challenge by a chimeric vaccine formulated with the Chlamydia muridarum major outer membrane protein variable domains using the Neisseria lactamica porin B as a scaffold

Chlamydia trachomatis is the most frequently detected sexually transmitted bacterial pathogen in the world. Attempts to control these infections with screening programs and antibiotics have failed and, therefore, a vaccine is the best approach to control this epidemic. The Chlamydia major outer membrane protein (MOMP) is the most protective subunit vaccine so far tested. Protection induced by MOMP is, in part, dependent on its tertiary structure. We have previously described new recombinant antigens composed of the Neisseria lactamica PorB engineered to express the variable domains (VD) from Chlamydia muridarum MOMP. Here we tested antigens containing each individual MOMP VD and different VD combinations. Following immunization, mice were challenged intranasally with C. muridarum. Our results show that three constructs, PorB/VD1–3, PorB/VD1–4, and PorB/VD1–2–4, elicited high serum IgG titers in vivo, significant IFN-γ levels upon T cells re-stimulation in vitro, and evidence of protective immunity in vivo. PorB/VD1–3, PorB/VD1–4, and PorB/VD1–2–4 immunized mice lost less body weight, had lighter lungs, and decreased numbers of inclusion forming units (IFUs) in lungs than other PorB/VD construct tested and mock PBS-immunized mice. These results suggest that this approach may be a promising alternative to the use of MOMP in a Chlamydia vaccine.

Long-lasting protective immunity against H7N9 infection is induced by intramuscular or CpG-adjuvanted intranasal immunization with the split H7N9 vaccine

There is an urgent need for efficient vaccines against the highly pathogenic avian influenza A viral strain H7N9. The duration and intensity of the immune response to H7N9 critically impacts the epidemiology of influenza viral infection at the population level. However, the insufficient immunogenicity of H7N9 raises concerns about vaccine efficacy. In this study, we evaluated the impact of immunization routes and the adjuvant CpG on the immune response to a split H7N9 vaccine in mice. Determination of humoral and cellular responses to the vaccine revealed that after four vaccine doses, high titers of H7N9-specific serum IgG, determined by the influenza hemagglutination inhibition (HI) assay, were induced through the intramuscular (i.m.) route and lasted for at least 40 weeks. CpG-adjuvanted immunization increased the levels of long-lived IFN-γ+ T cells and raised the Th1-biased IgG2a/IgG1 response ratio. In addition, aside from mucosal IgA, CpG-adjuvanted intranasal (i.n.) immunization elicited serum IgG and cellular responses of a similar duration and intensity to CpG-adjuvanted i.m. immunization. Mouse challenge assays demonstrated that 24 weeks following i.m. immunization without CpG or CpG-adjuvanted immunization through the i.m. or i.n. routes, both offered a high level of protection against H7N9 infection. These results indicate that efficient long-term protection against H7N9 can be achieved via the optimization of vaccination strategies, such as immunization doses, routes, and adjuvants.

UTI patients have pre-existing antigen-specific antibody titers against UTI vaccine antigens

Urinary tract infection (UTI) is most frequently caused by uropathogenic Escherichia coli (UPEC). Our laboratory has been developing an experimental vaccine targeting four UPEC outer membrane receptors involved in iron acquisition – IreA, FyuA, IutA, and Hma – to elicit protection against UTI. These vaccine targets are all expressed in humans during UTI. In the murine model, high titers of antigen-specific serum IgG or bladder IgA correlate with protection against transurethral challenge with UPEC. Our aim was to measure levels of pre-existing serum antibodies to UTI vaccine antigens in our target population. To accomplish this, we obtained sera from 64 consenting female patients attending a clinic for symptoms of cystitis. As a control, we also collected sera from 20 healthy adult male donors with no history of UTI. Total IgG and antigen-specific IgG titers were measured by ELISA. Of the 64 female patients, 29 had significant bacteriuria (>104 cfu/ml urine) and uropathogenic E. coli (UPEC). Thirty-five patients had non-significant bacteriuria (<104 cfu/ml). Antigen-specific IgG titers did not correlate with the presence or absence of the gene encoding the antigen in the infecting strain (when present), but rather titers were proportional to prevalence of genes encoding antigens among representative collections of UPEC isolates. Surprisingly, we obtained similar results when sera from healthy male patients without history of UTI were tested. Thus, unvaccinated adults have non-protective levels of pre-existing antibodies to UTI vaccine antigens, establishing an important baseline for our target population. This suggests that a UTI vaccine would need to boost pre-existing humoral responses beyond these background levels to protect from infection.

Haemophilus influenzae-protein D specific antibody correlate with protection against acute otitis media in young children

BackgroundHaemophilus influenzae (Hi) causes respiratory infections and pathogenesis of this microbe begins in the human nasopharynx (NP). The objective of this study was to assess the correlation of NP colonization-induced serum antibody levels to Hi protein D with risk of acute otitis media (AOM) in children <2 yr. Methods455 sera from 213 children (age 6–24 months old) were collected when they were colonized with Hi and when the children developed AOM. Presence of Hi during AOM was confirmed by culture of middle ear fluid. Quantitative ELISA was used to determine serum IgG against protein D antigen. ResultsAsymptomatic Hi NP colonization reduced the risk of future AOM infections. Higher serum IgG titers against Hi protein D were correlated with reduced future AOM risk. ConclusionColonization by Hi reduces future AOM risk. Higher antibody levels against protein D correlates with lower risk of AOM caused by Hi.

Overexpression and oral immunogenicity of a dengue antigen transiently expressed in Nicotiana benthamiana

The feasibility of a safe and cost-effective oral dengue vaccine approach was investigated. Consensus domain III of the dengue virus (DENV) serotypes 1–4 envelope glycoprotein was fused with cholera toxin B subunit (CTB–cEDIII) and cloned into TMV-based 3′ pro-module for high level expression. The construct was co-infiltrated with TMV-based 5′ pro-module and Integrase module into Nicotiana benthamiana leaves using vacuum agroinfiltration. CTB–cEDIII fusion protein directed to endoplasmic reticulum by C-terminal SEKEDL sequence was expressed at up to 8.4% of total soluble protein at 5 days-post infiltration (dpi). After oral immunization of mice with the transiently expressed plant-derived CTB–cEDIII, high titers of serum IgG and sIgA against both cEDIII and CTB were observed, peaking at week four. Sera from immunized mice reacted with each of the four DENV serotype-specific envelope proteins domain III. These results show that (i) the TMV-based pro-vector system is suitable for high-level expression of antigen in plant cells and (ii) the CTB fusion with cEDIII could be exploited as an oral vaccine candidate for induction of both systemic and mucosal immune responses to DENV.

Oral Administration of a Seed-based Bivalent Rotavirus Vaccine Containing VP6 and NSP4 Induces Specific Immune Responses in Mice.

Rotavirus is the leading cause of severe diarrheal disease among newborns. Plant-based rotavirus vaccines have been developed in recent years and have been proven to be effective in animal models. In the present study, we report a bivalent vaccine candidate expressing rotavirus subunits VP6 and NSP4 fused with the adjuvant subunit B of E. coli heat-labile enterotoxin (LTB) in maize seeds. The RT-PCR and Western blot results showed that VP6 and LTB-NSP4 antigens were expressed and accumulated in maize seeds. The expression levels were as high as 0.35 and 0.20% of the total soluble protein for VP6 and LTB-NSP4, respectively. Oral administration of transgenic maize seeds successfully stimulated systemic and mucosal responses, with high titers of serum IgG and mucosal IgA antibodies, even after long-term storage. This study is the first to use maize seeds as efficient generators for the development of a bivalent vaccine against rotavirus.

Maternal vaccination with a fimbrial tip adhesin and passive protection of neonatal mice against lethal human enterotoxigenic Escherichia coli challenge.

Globally, enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood and travelers' diarrhea, for which an effective vaccine is needed. Prevalent intestinal colonization factors (CFs) such as CFA/I fimbriae and heat-labile enterotoxin (LT) are important virulence factors and protective antigens. We tested the hypothesis that donor strand-complemented CfaE (dscCfaE), a stabilized form of the CFA/I fimbrial tip adhesin, is a protective antigen, using a lethal neonatal mouse ETEC challenge model and passive dam vaccination. For CFA/I-ETEC strain H10407, which has been extensively studied in volunteers, an inoculum of 2 × 10(7) bacteria resulted in 50% lethal doses (LD50) in neonatal DBA/2 mice. Vaccination of female DBA/2 mice with CFA/I fimbriae or dscCfaE, each given with a genetically attenuated LT adjuvant (LTK63) by intranasal or orogastric delivery, induced high antigen-specific serum IgG and fecal IgA titers and detectable milk IgA responses. Neonates born to and suckled by dams antenatally vaccinated with each of these four regimens showed 78 to 93% survival after a 20× LD50 challenge with H10407, compared to 100% mortality in pups from dams vaccinated with sham vaccine or LTK63 only. Crossover experiments showed that high pup survival rates after ETEC challenge were associated with suckling but not birthing from vaccinated dams, suggesting that vaccine-specific milk antibodies are protective. In corroboration, preincubation of the ETEC inoculum with antiadhesin and antifimbrial bovine colostral antibodies conferred a dose-dependent increase in pup survival after challenge. These findings indicate that the dscCfaE fimbrial tip adhesin serves as a protective passive vaccine antigen in this small animal model and merits further evaluation.

Physiopathological effects of Escherichia coli O157:H7 inoculation in weaned calves fed with colostrum containing antibodies to EspB and Intimin

Escherichia coli O157:H7 is responsible for severe intestinal disease and hemolytic uremic syndrome (HUS), a serious systemic complication which particularly affects children. Cattle are the primary reservoir for E. coli O157:H7 and the main source of infection for humans. In this study, we evaluated the ability of transferred maternal colostral antibodies against γ-Intimin C280 and EspB, to protect young weaned calves from E. coli O157:H7 infection. Hyperimmune colostra were obtained by immunization of pregnant cows with a mix of the mentioned antigens. All vaccinated cows mounted a significant IgG response against γ-Intimin C280, and EspB in sera and colostra. Colostrum-fed calves also exhibited high serum IgG titers against γ-Intimin C280 and EspB along with a rise in mucosal γ-Intimin C280-specific IgG antibodies at recto-anal junction and ileum. Additionally, 70 day-old calves received a challenge with E. coli O157:H7 but no reduction in total bacterial shedding or frequency of E. coli O157:H7 excretion from these calves was observed. Most tissue samples showed granulocyte focal infiltrations of the lamina propria and enterocyte erosion. In conclusion, up to the 70th day, the passively acquired γ-Intimin-C280 and EspB-IgG antibodies present in sera and recto-anal mucosa reached a titer insufficient to reduce EHEC O157 shedding and damages of experimentally inoculated young calves.

Immunogenicity of a Prime-Boost Vaccine Containing the Circumsporozoite Proteins of Plasmodium vivax in Rodents

Plasmodium vivax is the most widespread and the second most prevalent malaria-causing species in the world. Current measures used to control the transmission of this disease would benefit from the development of an efficacious vaccine. In the case of the deadly parasite P. falciparum, the recombinant RTS,S vaccine containing the circumsporozoite antigen (CSP) consistently protects 30 to 50% of human volunteers against infection and is undergoing phase III clinical trials in Africa with similar efficacy. These findings encouraged us to develop a P. vivax vaccine containing the three circulating allelic forms of P. vivax CSP. Toward this goal, we generated three recombinant bacterial proteins representing the CSP alleles, as well as a hybrid polypeptide called PvCSP-All-CSP-epitopes. This hybrid contains the conserved N and C termini of P. vivax CSP and the three variant repeat domains in tandem. We also generated simian and human recombinant replication-defective adenovirus vectors expressing PvCSP-All-CSP-epitopes. Mice immunized with the mixture of recombinant proteins in a formulation containing the adjuvant poly(I·C) developed high and long-lasting serum IgG titers comparable to those elicited by proteins emulsified in complete Freund's adjuvant. Antibody titers were similar in mice immunized with homologous (protein-protein) and heterologous (adenovirus-protein) vaccine regimens. The antibodies recognized the three allelic forms of CSP, reacted to the repeated and nonrepeated regions of CSP, and recognized sporozoites expressing the alleles VK210 and VK247. The vaccine formulations described in this work should be useful for the further development of an anti-P. vivax vaccine.

Immunologic characterization of a novel inactivated nasal mumps virus vaccine adjuvanted with Protollin

An inactivated, mucosal mumps virus (MuV) vaccine would address many of the problems associated with current live-attenuated formulations. Protollin (Prl)-based adjuvants (containing TLR2 and TLR4 ligands) are well-suited for nasal administration. We sought to develop an inactivated whole-virus nasal vaccine for MuV using the Prl adjuvant/delivery vehicle and to test tolerability and immunogenicity in a mouse model. BALB/c mice exhibited signs of transient reactogenicity (hunched posture, erect fur, weight loss ≤10% of total body weight) following administration of intranasal MuV-Prl vaccines, though most of these manifestations resolved within 24h. Compared to high-dose unadjuvanted vaccine (8μgMuV), administration of high-dose adjuvanted formulation (8μgMuV-Prl) induced greater MuV-specific serum IgG (3.26E6ng/mL vs. 2.2E5ng/mL, 8μgMuV-Prl vs. 8μgMuV, p<0.001) and mucosal IgA (128ng/mL vs. 45ng/mL, 8μgMuV-Prl vs. 8μgMuV, p<0.05). Serum IgG isotypes and splenocyte cytokine secretion induced by MuV-Prl suggested a predominant T helper cell (Th)1-type immune response. This response was characterized by: (1) ≥four-fold increase of IgG2a levels compared to IgG1; and (2) high IL-2 (644pg/mL)/IFN-γ (228pg/mL) and low IL-5 (31pg/mL) secretion in MuV-restimulated splenocytes from animals receiving MuV-Prl formulations. MuV-Prl vaccination induced higher levels of serum antibodies capable of neutralizing MuV in vitro than MuV alone, particularly for high-dose 8μg formulations (357 neutralizing units (NU)/mL vs. 32NU/mL, 8μgMuV-Prl vs. 8μgMuV, p<0.001). Thus, nasal MuV-Prl vaccines are fairly well-tolerated and highly immunogenic in mice.

Human Rotavirus-Specific IgM Memory B Cells Have Differential Cloning Efficiencies and Switch Capacities and Play a Role in Antiviral ImmunityIn Vivo

Protective immunity to rotavirus (RV) is primarily mediated by antibodies produced by RV-specific memory B cells (RV-mBc). Of note, most of these cells express IgM, but the function of this subset is poorly understood. Here, using limiting dilution assays of highly sort-purified human IgM(+) mBc, we found that 62% and 21% of total (non-antigen-specific) IgM(+) and RV-IgM(+) mBc, respectively, switched in vitro to IgG production after polyclonal stimulation. Moreover, in these assays, the median cloning efficiencies of total IgM(+) (17%) and RV-IgM(+) (7%) mBc were lower than those of the corresponding switched (IgG(+) IgA(+)) total (34%) and RV-mBc (17%), leading to an underestimate of their actual frequency. In order to evaluate the in vivo role of IgM(+) RV-mBc in antiviral immunity, NOD/Shi-scid interleukin-2 receptor-deficient (IL-2Rγ(null)) immunodeficient mice were adoptively transferred highly purified human IgM(+) mBc and infected with virulent murine rotavirus. These mice developed high titers of serum human RV-IgM and IgG and had significantly lower levels than control mice of both antigenemia and viremia. Finally, we determined that human RV-IgM(+) mBc are phenotypically diverse and significantly enriched in the IgM(hi) IgD(low) subset. Thus, RV-IgM(+) mBc are heterogeneous, occur more frequently than estimated by traditional limiting dilution analysis, have the capacity to switch Ig class in vitro as well as in vivo, and can mediate systemic antiviral immunity.

The mucosal adjuvant potential of cross-linked dextran microspheres as dry powder.

The immunoadjuvant potential of cross-linked dextran microspheres (CDM) as absorption enhancer and Quillaja saponins (QS) as immunomodulator adjuvant was evaluated. CDM loaded or tetanus-mixed toxoid (TT) or Quillaja saponin (QS) were nasally administered to rabbits in dry powder form, three times in 2 weeks interval and serum IgG and nasal lavage sIgA titers were determined by ELISA. The highest serum IgG titer was induced by parenteral immunization through alum adsorbed TT (P< 0.001). Among nasally immunized groups, the highest serum IgG titer was induced by (TT+QS)CDM (P< 0.01). Mixing of CDM with TT+QS powders (CDM+TT+QS), could not induce the high serum IgG titers as (TT+QS)CDM (P< 0.01). CDM loaded with TT+QS induced higher IgG titers than CDM loaded with TT alone (P< 0.01). No significant difference was observed in nasal lavage sIgA titers of various groups. CDM microspheres loaded with TT+QS significantly increased serum anti-TT IgG titers, but mixing of CDM with TT+QS powder could not increase IgG titers. Both QS and CDM adjuvant could not significantly increase the lavage anti-TT IgA titers.

Sublingual vaccination with fusion protein consisting of the functional domain of hemagglutinin A of Porphyromonas gingivalis and Escherichia coli maltose-binding protein elicits protective immunity in the oral cavity

This study demonstrated that sublingual immunization with a fusion protein, 25k-hagA-MBP, which consists of a 25-kDa antigenic region of hemagglutinin A purified from Porphyromonas gingivalis fused to maltose-binding protein (MBP) originating from Escherichia coli as an adjuvant, elicited protective immune responses. Immunization with 25k-hagA-MBP induced high levels of antigen-specific serum IgG and IgA, as well as salivary IgA. High level titers of serum IgG and IgA were also induced for almost 1 year. In an IgG subclass analysis, sublingual immunization with 25k-hagA-MBP induced both IgG1 and IgG2b antibody responses. Additionally, numerous antigen-specific IgA antibody-forming cells were detected from the salivary gland 7 days after the final immunization. Mononuclear cells isolated from submandibular lymph nodes (SMLs) showed significant levels of proliferation upon restimulation with 25k-hagA-MBP. An analysis of cytokine responses showed that antigen-specific mononuclear cells isolated from SMLs produced significantly high levels of IL-4, IFN-γ, and TGF-β. These results indicate that sublingual immunization with 25k-hagA-MBP induces efficient protective immunity against P. gingivalis infection in the oral cavity via Th1-type and Th2-type cytokine production.