Serum Neutralizing Antibody Levels Research Articles

Protective efficacy and immune responses of largemouth bass (Micropterus salmoides) immunized with an inactivated vaccine against the viral hemorrhagic septicemia virus genotype IVa

Viral hemorrhagic septicemia virus (VHSV) poses a significant threat to the aquaculture industry, prompting the need for effective preventive measures. Here, we developed an inactivated VHSV and revealed the molecular mechanisms underlying the host's protective response against VHSV. The vaccine was created by treating VHSV with 0.05 % formalin at 16 °C for 48 h, which was determined to be the most effective inactivation method. Compared with nonvaccinated fish, vaccinated fish exhibited a remarkable increase in survival rate (99 %) and elevated levels of serum neutralizing antibodies, indicating strong immunization. To investigate the gene changes induced by vaccination, RNA sequencing was performed on spleen samples from control and vaccinated fish 14 days after vaccination. The analysis revealed 893 differentially expressed genes (DEGs), with notable up-regulation of immune-related genes such as annexin A1a, coxsackievirus and adenovirus receptor homolog, V-set domain-containing T-cell activation inhibitor 1-like, and heat shock protein 90 alpha class A member 1 tandem duplicate 2, indicating a vigorous innate immune response. Furthermore, KEGG enrichment analysis highlighted significant enrichment of DEGs in processes related to antigen processing and presentation, necroptosis, and viral carcinogenesis. GO enrichment analysis further revealed enrichment of DEGs related to the regulation of type I interferon (IFN) production, type I IFN production, and negative regulation of viral processes. Moreover, protein-protein interaction network analysis identified central hub genes, including IRF3 and HSP90AA1.2, suggesting their crucial roles in coordinating the immune response elicited by the vaccine. These findings not only confirm the effectiveness of our vaccine formulation but also offer valuable insights into the underlying immunological mechanisms, which can be valuable for future vaccine development and disease management in the aquaculture industry.

Comparison of Omicron breakthrough infection versus monovalent SARS-CoV-2 intramuscular booster reveals differences in mucosal and systemic humoral immunity

Our understanding of the quality of cellular and humoral immunity conferred by coronavirus disease 2019 (COVID-19) vaccination alone versus vaccination plus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) breakthrough (BT) infection remains incomplete. While the current (2023) SARS-CoV-2 immune landscape of Canadians is complex, in late 2021 most Canadians had either just received a third dose of COVID-19 vaccine, or had received their two dose primary series and then experienced an Omicron BT. Herein we took advantage of this coincident timing to contrast cellular and humoral immunity conferred by three doses of vaccine versus two doses plus BT. Our results show that mild BT infection induces cell-mediated immune responses to variants comparable to an intramuscular vaccine booster dose. In contrast, BT subjects had higher salivary IgG and IgA levels against the Omicron spike and enhanced reactivity to the ancestral spike for the IgA isotype, which also reacted with SARS-CoV-1. Serum neutralizing antibody levels against the ancestral strain and the variants were also higher after BT infection. Our results support the need for vaccines that emulate the enhanced mucosal and humoral immunity induced by Omicron BT without exposing individuals to the risks associated with SARS-CoV-2 infection.One Sentence SummaryOmicron breakthrough elicits cross-reactive systemic and mucosal immune responses in vaccinated adults.

Preparation and immunoprotective effects of a virus-like particle candidate vaccine of the dominant epidemic D3 genotype coxsackievirus A6 in China

Coxsackievirus A6 of the D3a genotype (CVA6 D3a) is a primary pathogen causingmainland of China's hand, foot, and mouth disease (HFMD). Viral-like particle (VLP) vaccines represent a potential candidate vaccine to prevent HFMD. This study collected Anti-CVA6 D3a VLPs serum from BALB/c female mice immunized using CVA6 D3a VLPs. The neutralizing antibody levels were compared against the representative 14-JX2018 (D3a) and N4-YN2015 (D3b) strains between the antisera of different immune pathways. The immunoprotective effect of anti-CVA6 D3a VLPs against these strains was monitored using pathological sections and immunohistochemical results of lung and skeletal muscle tissues in seven-day-old Institute of Cancer Research (ICR) mice. Immunological protection against different branches of viruses was evaluated in 7-day-old (serum passive immune protection) and 14-day-old (VLPs active immune protection) neonatal ICR mice models. Serum-neutralizing antibody levels were positively correlated with the number of immunizations and higher against 14-JX2018 than against N4-YN2015. Furthermore, these levels were significantly higher with abdominal injection than intramuscular injection. The immunized serum of 7-day-old ICR mice inoculated three times was 100 % protected against CVA6 D3a 14-JX2018 (lethal titer: 106.25 TCID50) and CVA6 D3b N4-YN2015 (lethal titer: 105.25TCID50) fatal attacks, respectively. For ICR mice that have completed two active immunizations for 14 days, both CVA6 D3a 14-JX2015 (challenge titer: 108.25 TCID50) and CVA6 D3b N4-YN2015 (challenge titer: 107.25 TCID50) were used for the challenge, and the mice were able to survive. Overall, the CVA6 D3a VLPs prepared in this study are a potential vaccine candidate for CVA6, as it has the optimal protective effect against both CVA6 D3a and D3b evolutionary branches viruses.

Intranasal boosting with RBD-HR protein vaccine elicits robust mucosal and systemic immune responses

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has decreased the efficacy of SARS-CoV-2 vaccines in containing coronavirus disease 2019 (COVID-19) over time, and booster vaccination strategies are urgently necessitated to achieve sufficient protection. Intranasal immunization can improve mucosal immunity, offering protection against the infection and sustaining the spread of SARS-CoV-2. In this study, an intranasal booster of the RBD-HR vaccine after two doses of the mRNA vaccine significantly increased the levels of specific binding antibodies in serum, nasal lavage fluid, and bronchoalveolar lavage fluid compared with only two doses of mRNA vaccine. After intranasal boosting with the RBD-HR vaccine, the levels of serum neutralizing antibodies against prototype and variant strains of SARS-CoV-2 pseudoviruses were markedly higher than those in mice receiving mRNA vaccine alone, and intranasal boosting with the RBD-HR vaccine also inhibited the binding of RBD to hACE2 receptors. Furthermore, the heterologous intranasal immunization regimen promoted extensive memory T cell responses and activated CD103+ dendritic cells in the respiratory mucosa, and potently enhanced the formation of T follicular helper cells and germinal center B cells in vital immune organs, including mediastinal lymph nodes, inguinal lymph nodes, and spleen. Collectively, these data infer that heterologous intranasal boosting with the RBD-HR vaccine elicited broad protective immunity against SARS-CoV-2 both locally and systemically.

Influenza H3 hemagglutinin vaccine with scrambled immunodominant epitopes elicits antibodies directed toward immunosubdominant head epitopes.

Vaccination is the most effective countermeasure to reduce the severity of influenza. Current seasonal influenza vaccines mainly elicit humoral immunity targeting hemagglutinin (HA). In particular, the amino acid residues around the receptor-binding site in the HA head domain are predominantly targeted by humoral immunity as "immunodominant" epitopes. However, mutations readily accumulate in the head domain due to high plasticity, resulting in antigenic drift and vaccine mismatch, particularly with influenza A (H3N2) viruses. A vaccine strategy that targets more conserved immunosubdominant epitopes is required to attain a universal vaccine. Here, we designed an H3 HA vaccine antigen with various amino acids at immunodominant epitopes of the HA head domain, termed scrambled HA (scrHA). In ferrets, scrHA vaccination induced lower serum neutralizing antibody levels against homologous virus compared with wild-type (WT) HA vaccination; however, similar levels of moderately neutralizing titers against antigenically distinct H3N2 viruses were observed. Ferrets vaccinated with scrHA twice and then challenged with homologous or heterologous virus showed the same level of reduced virus shedding in nasal swabs as WT HA-vaccinated animals but reduced body temperature increase, whereas WT HA-vaccinated ferrets exhibited body temperature increases similar to those of mock-vaccinated animals. scrHA elicited antibodies against HA immunodominant and -subdominant epitopes at lower and higher levels, respectively, than WT HA vaccination, whereas antistalk antibodies were induced at the same level for both groups, suggesting scrHA-induced redirection from immunodominant to immunosubdominant head epitopes. scrHA vaccination thus induced broader coverage than WT HA vaccination by diluting out the immunodominancy of HA head epitopes. IMPORTANCE Current influenza vaccines mainly elicit antibodies that target the immunodominant head domain, where strain-specific mutations rapidly accumulate, resulting in frequent antigenic drift and vaccine mismatch. Targeting conserved immunosubdominant epitopes is essential to attain a universal vaccine. Our findings with the scrHA developed in this study suggest that designing vaccine antigens that "dilute out" the immunodominancy of the head epitopes may be an effective strategy to induce conserved immunosubdominant epitope-based immune responses.

Omicron Breakthrough Infection Elicits Superior Humoral and Mucosal Immunity to SARS-CoV-2 Variants than Booster Doses

Abstract In light of ongoing booster vaccination campaigns, one recurring question is whether individuals infected with SARS-CoV-2 should still receive a booster dose if they were infected after completing their primary series. We aimed to assess the intensity of functional humoral and cellular immune responses induced by breakthrough infections during the B.1.1.529 wave in Canada compared to individuals who received three doses of vaccine. Because hybrid immunity has been shown to induce particularly potent immunity, we hypothesized that breakthrough infections could induce superior immune response to variants than booster doses. To address this important public health question, we analyzed samples from two cohorts of individuals naïve to SARS-CoV-2 or who were primary infected before vaccination. We compared responses 45 days after a third vaccine dose or an Omicron breakthrough infection. Our results show that mild breakthrough infection induces cell-mediated immune response to variants comparable to a vaccine booster dose. In contrast, breakthrough subjects had higher salivary IgG and IgA levels against the Omicron Spike and enhanced reactivity to the ancestral Spike for the IgA isotype, which also reacted with SARS-CoV-1. Serum neutralizing antibody levels against the ancestral strain and the variants were also higher after breakthrough infection, especially in individuals naïve to the infection prior to vaccination. Altogether our results demonstrate that an Omicron breakthrough infection can substitute a booster dose of vaccine, while also supporting the need for mucosal vaccine boosts to potentially replicate the improvement on mucosal and humoral immunity without the risks associated to being infected with SARS-CoV-2. Research supported by grants from the Canadian Institutes of Health Research, Public Health Agency of Canada and the Ontario COVID-19 Rapid Research Fund through Ontario Together.

Impact of maternal and pre-existing antibodies on immunogenicity of inactivated rotavirus vaccines

Rotavirus (RV) is a major pathogen causing severe diarrhea in infants and children aged less than 5 years. Vaccination is an economically feasible and effective strategy to prevent rotavirus infections. However, immune efficacy of live vaccines could be interfered by maternal antibodies and pre-existing antibodies of children. To develop an inactivated rotavirus vaccine (IRV), we had previously isolated a wild-type human rotavirus strain ZTR-68-A (G1P[8]) from the fecal samples of infants having severe diarrhea in a region endemic for the presence of this pathogen. In our present study, we assessed whether the presence of maternal and pre-existing antibodies in newborn BALB/c mice affected the immunogenicity of IRV administered to these animals. Our results indicate that maternal antibodies, generated from either vaccine immunization or rotavirus infection, showed partial influence with the immune responses generated by two doses of IRV vaccination. Increasing the number of immunizations can significantly improve the titer of serum neutralizing antibody and a seroconversion rate of up to 100%. In newborn mice, single-virus infection did not elicit detectable levels of serum neutralizing antibodies. After an IRV vaccination, the immune responses of these mice remained unaffected, with no significant differences in titers compared with those of control-group mice. In summary, choosing a suitable immunization dose and dosing frequency is essential for the immune effectiveness of IRV. The results of this study will provide animal experimental support for the IRV clinical research in future.

Covax-19/Spikogen® vaccine based on recombinant spike protein extracellular domain with Advax-CpG55.2 adjuvant provides single dose protection against SARS-CoV-2 infection in hamsters

COVID-19 presents an ongoing global health crisis. Protein-based COVID-19 vaccines that are well-tolerated, safe, highly-protective and convenient to manufacture remain of major interest. We therefore sought to compare the immunogenicity and protective efficacy of a number of recombinant SARS-CoV-2 spike protein candidates expressed in insect cells. By comparison to a full length (FL) spike protein detergent-extracted nanoparticle antigen, the soluble secreted spike protein extracellular domain (ECD) generated higher protein yields per liter of culture and when formulated with either Alum-CpG55.2 or Advax-CpG55.2 combination adjuvants elicited robust antigen-specific humoral and cellular immunity in mice. In hamsters, the spike ECD when formulated with either adjuvant induced high serum neutralizing antibody titers even after a single dose. When challenged with the homologous SARS-CoV-2 virus, hamsters immunized with the adjuvanted spike ECD exhibited reduced viral load in day 1–3 oropharyngeal swabs and day 3 nasal turbinate tissue and had no recoverable infectious virus in day 3 lung tissue. The reduction in lung viral load correlated with less weight loss and lower lung pathology scores. The formulations of spike ECD with Alum-CpG55.2 or Advax-CpG55.2 were protective even after just a single dose, although the 2-dose regimen performed better overall and required only half the total amount of antigen. Pre-challenge serum neutralizing antibody levels showed a strong correlation with lung protection, with a weaker correlation seen with nasal or oropharyngeal protection. This suggests that serum neutralizing antibody levels may correlate more closely with systemic, rather than mucosal, protection. The spike protein ECD with Advax-CpG55.2 formulation (Covax-19® vaccine) was selected for human clinical development.

A porcine reproductive and respiratory syndrome virus (PRRSV)-specific IgM as a novel adjuvant for an inactivated PRRSV vaccine improves protection efficiency and enhances cell-mediated immunity against heterologous PRRSV challenge

Current strategies for porcine reproductive and respiratory syndrome (PRRS) control are inadequate and mainly restricted to immunization using different PRRS virus (PPRSV) vaccines. Although there are no safety concerns, the poor performance of inactivated PRRSV vaccines has restricted their practical application. In this research, we employed the novel PRRSV-specific IgM monoclonal antibody (Mab)-PR5nf1 as a vaccine adjuvant for the formulation of a cocktail composed of inactivated PRRSV (KIV) and Mab-PR5nf1 along with a normal adjuvant to enhance PRRSV-KIV vaccine-mediated protection and further compared it with a normal KIV vaccine and modified live virus vaccine (MLV). After challenge with highly pathogenic (HP)-PRRSV, our results suggested that the overall survival rate (OSR) and cell-mediated immunity (CMI), as determined by serum IFN-γ quantification and IFN-γ ELISpot assay, were significantly improved by adding PRRSV-specific IgM to the PRRSV-KIV vaccine. It was also notable that both the OSR and CMI in the Mab-PR5nf1-adjuvanted KIV group were even higher than those in the MLV group, whereas the CMI response is normally poorly evoked by KIV vaccines or subunit vaccines. Compared with those in piglets immunized with the normal KIV vaccine, viral shedding and serum neutralizing antibody levels were also improved, and reduced viral shedding appeared to be a result of enhanced CMI caused by the inclusion of IgM as an adjuvant. In conclusion, our data provide not only a new formula for the development of an effective PRRSV-KIV vaccine for practical use but also a novel method for improving antigen-specific CMI induction by inactivated vaccines and subunit vaccines.

Safety and immunogenicity evaluation of inactivated whole-virus-SARS-COV-2 as emerging vaccine development in Egypt.

BackgroundCurrent worldwide pandemic coronavirus disease 2019 (COVID-19) with high numbers of mortality rates and huge economic problems require an urgent demand for safe and effective vaccine development. Inactivated SARS-CoV2 vaccine with alum. Hydroxide can play an important role in reducing the impacts of the COVID-19 pandemic. In this study, vaccine efficacy was evaluated through the detection of the neutralizing antibodies that protect mice from challenge with SARS-CoV 2 3 weeks after the second dose. We conclude that the vaccine described here has safety and desirable properties, and our data support further development and plans for clinical trials.MethodsCharacterized SARS-COV-2 strain, severe acute respiratory syndrome coronavirus 2 isolates (SARS-CoV-2/human/EGY/Egy-SERVAC/2020) with accession numbers; MT981440; MT981439; MT981441; MT974071; MT974069; and MW250352 at GenBank were isolated from Egyptian patients SARS-CoV-2-positive. Development of inactivated vaccine was carried out in a BSL-3 facilities and the immunogenicity was determined in mice at two doses (55 and 100 μg per dose).ResultsThe distinct cytopathic effect induced by SARS-COV-2 propagation on Vero cell monolayers and the viral particles were identified as Coronaviridae by transmission electron microscopy and RT-PCR on infected cells cultures. Immunogenicity of the developed vaccine indicated the high antigen-binding and neutralizing antibody titers, regardless of the dose concentration, with excellent safety profiles and no deaths or clinical symptoms in mice groups. The efficacy of the inactivated vaccine formulation was tested by the wild virus challenge of the vaccinated mice and viral replication detection in lung tissues.ConclusionsVaccinated mice recorded complete protection from challenge infection via inhibition of SARS-COV-2 replication in the lung tissues of mice following virus challenge, regardless of the level of serum neutralizing antibodies. This finding will support future trials for the evaluation of an applicable SARS-CoV-2 vaccine candidate.

A purified inactivated vaccine derived from Vero cell-adapted zika virus elicits protection in mice

The Zika virus (ZIKV) outbreak in 2015–2016 raised public health concerns and created a pressing need for vaccine development. However, no vaccine has been developed and most of the ones under development use a single serotype of ZIKV. In this study, we established a Vero cell-adapted ZIKV strain (GMZ-002) and developed a purified inactivated virus (PIV) vaccine. GMZ-002 presented significantly increased productivity in Vero cells, and IFNAR1-blocked C57BL/6 mice administered two doses of the PIV were fully protected against lethal challenge. Vaccine efficacy was illustrated by the high level of serum neutralizing antibodies and strong innate immune response, along with an absence of detectable viremia in vaccinated mice. Furthermore, anti-sera neutralized both African and Asian genetic lineages of the virus in vitro. Our results suggest that GMZ-002 PIV elicited robust and persistent protective immunity, and therefore represents a promising vaccine candidate for ZIKV.

Single dose of a replication-defective vaccinia virus expressing Zika virus-like particles is protective in mice

Zika virus (ZIKV), a flavivirus transmitted primarily by infected mosquitos, can cause neurological symptoms such as Guillian–Barré syndrome and microcephaly. We developed several vaccinia virus (VACV) vaccine candidates for ZIKV based on replication-inducible VACVs (vINDs) expressing ZIKV pre-membrane (prM) and envelope (E) proteins (vIND-ZIKVs). These vIND-ZIKVs contain elements of the tetracycline operon and replicate only in the presence of tetracyclines. The pool of vaccine candidates was narrowed to one vIND-ZIKV containing a novel mutation in the signal peptide of prM that led to higher expression and secretion of E and production of virus-like particles, which was then tested for safety, immunogenicity, and efficacy in mice. vIND-ZIKV grows to high titers in vitro in the presence of doxycycline (DOX) but is replication-defective in vivo in the absence of DOX, causing no weight loss in mice. C57BL/6 mice vaccinated once with vIND-ZIKV in the absence of DOX (as a replication-defective virus) developed robust levels of E-peptide-specific IFN-γ-secreting splenocytes and anti-E IgG titers, with modest levels of serum-neutralizing antibodies. Vaccinated mice treated with anti-IFNAR1 antibody were completely protected from ZIKV viremia post-challenge after a single dose of vIND-ZIKV. Furthermore, mice with prior immunity to VACV developed moderate anti-E IgG titers that increased after booster vaccination, and were protected from viremia only after two vaccinations with vIND-ZIKV.

Development of Improved Mumps Vaccine Candidates by Mutating Viral mRNA Cap Methyltransferase Sites in the Large Polymerase Protein.

Although a live attenuated vaccine is available for controlling mumps virus (MuV), mumps still outbreaks frequently worldwide. The attenuated MuV vaccine strain S79 is widely used in mumps vaccination in China, but still with many shortcomings, among which the most prominent are the side effects and decreased immunity. Therefore, there is a need to further improve the safety and efficacy of the current MuV vaccine. In the present study, we further attenuated MuV S79 vaccine strain by inhibiting viral mRNA methyltransferase (MTase). We generated a panel of eight recombinant MuVs (rMuVs) carrying mutations in the MTase catalytic site or S-adenosylmethionine (SAM) binding site in the large (L) polymerase protein. These rMuVs are genetically stable and seven rMuVs are more attenuated in replication in cell culture and five rMuVs are more attenuated in replication in lungs of cotton rats compared with the parental vaccine strain S79. Importantly, cotton rats vaccinated with these seven rMuV mutants produced high levels of serum neutralizing antibodies and were completely protected against challenge with a wild-type MuV strain (genotype F). Therefore, our results demonstrate that alteration in the MTase catalytic site or SAM binding site in MuV L protein improves the safety or the immunogenicity of the MuV vaccine and thus mRNA cap MTase may be an effective target for the development of new vaccine candidates for MuV.Electronic supplementary materialThe online version of this article (10.1007/s12250-020-00326-y) contains supplementary material, which is available to authorized users.

The role of viral particle integrity in the serological assessment of foot-and-mouth disease virus vaccine-induced immunity in swine.

The efficacy of foot-and-mouth disease virus (FMDV) inactivated vaccines is mainly dependent on the integrity of the whole (146S) viral particles. If the intact capsids disassemble to 12S subunits, antibodies against internal-not protective epitopes, may be induced. Serological correlates with protection may be hampered if antibodies against internal epitopes are measured. Here we compared the performance of different ELISAs with the virus-neutralization test (VNT) that measures antibodies against exposed epitopes. Sera from pigs immunized with one dose of an expired commercial FMDV vaccine were used. This vaccine contained about 50% of O1/Campos and over 90% of A24/Cruzeiro strains total antigen as whole 146S particles. Specific-total antibodies were measured with the standard liquid-phase blocking ELISA (LPBE). We also developed an indirect ELISA (IE) using sucrose gradient purified 146S particles as capture antigen to titrate total antibodies, IgM, IgG1 and IgG2. A good correlation was found between VNT titers and IgG-ELISAs for A24/Cruzeiro, with the lowest correlation coefficient estimated for IgG2 titers. For O1/Campos, however, the presence of antibodies against epitopes different from those of the whole capsid, elicited by the presence of 12S particles in the vaccine, hampered the correlation between LPBE and VNT, which was improved by using purified O1/Campos 146S-particles for the liquid-phase of the LPBE. Interestingly, 146S particles but not 12S were efficiently bound to the ELISA plates, confirming the efficiency of the IE to detect antibodies against exposed epitopes. Our results indicate that any serological test assessing total antibodies or IgG1 against epitopes exposed in intact 146S-particles correlate with the levels of serum neutralizing antibodies in vaccinated pigs, and might potentially replace the VNT, upon validation. We recommend that antigen used for serological assays aimed to measure protective antibodies against FMDV should be controlled to ensure the preservation of 146S viral particles.

Dietary cholesterol affects the pathogenesis of influenza A virus infection in mice

Abstract Influenza is one of the most prevalent and devastating infectious diseases in the world. But it is unclear how host factors may function to increase disease susceptibility or severity. Here, we sought to determine the role of dietary cholesterol in modulating the immune response or altering viral activity during influenza A infection. A pilot experiment indicated mice fed a 2% cholesterol diet prior to inoculation with mouse-adapted human influenza A virus (Puerto Rico/8/1934 H1N1; 1 HAU) exhibit greater morbidity compared to controls fed an energy density-matched diet. To confirm these data, 5-week-old C57BL/6 mice were fed either standard rodent diet or matched diet with 2% cholesterol for 5 weeks, then inoculated intranasally with either saline or flu (0.7 HAU). The effect of diet on circulating leukocytes was determined by flow cytometry. Serum cholesterol was measured by ELISA. Viral load and pathology were quantified by RTqPCR and immunohistochemistry. Finally, serum virus-neutralizing antibody levels were evaluated by haemagglutination inhibition. As observed previously, infected cholesterol-fed mice exhibited greater weight loss than diet matched controls. Total plasma cholesterol did not differ between diet groups. Infection increased the percentage of circulating granulocytes and decreased that of B cells at day 4 post-infection (p.i.). In contrast, diet did not affect circulating leukocytes. The amount of viral RNA at day 8 p.i. as well as serum neutralizing antibody levels were not affected by diet. Taken together, these data suggest dietary cholesterol affects the pathogenesis of influenza infection but prompt further study into better understanding the mechanism.

Testing of Ferarabivac anti-rabies live vaccine for wild carnivores for its immunogenicity and protectivity

Rabies is one of the most important human and animal viral diseases, being one of the most dangerous zoonoses, causing damage to the central nervous system with an inevitable fatal outcome. This disease is of global concern, and it attracts special attention of international organizations (WHO, OIE, FAO, GARC) and of veterinary services in many countries around the world. A variety of anti-rabies vaccines have been used for specific rabies prevention in wild carnivores, however, the safety and effectiveness of some of them is doubtful. New, more advanced products are being developed, one of which is Ferarabivac, a live oral vaccine. The vaccine was tested for its immunogenicity and protectivity in wild carnivores. The optimal immunizing dose was 2.0 cm3, with the infectivity titre of RV-97 strain of at least 6.00 lg KKID50/cm3. Anti-rabies antibody titres detected in the blood sera of foxes and raccoon dogs 14 days post vaccination, were 0.70 ± 0.18 and 0.73 ± 0.19 IU/cm3, respectively, which provided protection against rabies virus infection (≥ 0.50 IU/cm3). Rabies virus neutralizing antibodies in foxes reached their maximum level of 4.30 ± 0.32 IU/cm3 50 days post vaccination. Antibody titres in vaccinated raccoon dogs also reached their maximum level of 4.53 ± 0.27 IU/cm3 50 days post vaccination. The minimum protective threshold levels of serum neutralizing antibodies was determined 12 months after the vaccination, and it was 0.62 ± 0.28 and 0.71 ± 0.17 IU/cm3 in foxes and raccoon dogs, respectively, which proves the necessity to perform booster vaccination one year later. No animals vaccinated against rabies with Ferarabivac live vaccine showed any clinical signs of the disease during the entire observation period following the challenge test carried out 30 days post vaccination.

Prevalent levels of RSV serum neutralizing antibodies in healthy adults outside the RSV-season

ABSTRACT One of the main challenges in early clinical research with respiratory syncytial virus (RSV) live-attenuated vaccines (LAVs) is to assess immunogenicity in healthy adults. Healthy adults will have preexisting levels of serum neutralizing antibodies that could prematurely neutralize the LAV and underestimate the potential effect of the vaccine on the immune system. Data on prevalence and distribution of virus neutralizing titers (VNTs) in healthy adults is limited and there is no absolute threshold for protection against RSV-infection that can serve as an eligibility criterion in early phase trials. We assessed the RSV-specific serum VNT in healthy adults outside the Dutch RSV-Season in two clinical studies performed in 2017 (exploratory study, n = 100) and 2018 (first-in-human LAV-study, n = 190) using the same neutralizing assay. Our findings show that the prevalence and distribution of serum VNT was overall consistent in the two clinical studies. Log2 VNTs were normally distributed, distributions of VNTs were similar and there was no statistical difference in mean log2 VNT for both studies (p = .3). Serum VNTs were comparable during the 6 months of screening in the FIH LAV-study. Our findings will help to determine a cutoff serum VNT to be used as an eligibility criterion in future early phase clinical trials.

Persistent Dengue Infection in an Immunosuppressed Patient Reveals the Roles of Humoral and Cellular Immune Responses in Virus Clearance

Detailed understanding of the roles of humoral and cellular immune responses in sterilizing dengue virus (DENV) infection in humans is required to inform effective vaccine development. We report an unusual case of persistent DENV infection in a lymphopenic renal transplant recipient who was therapeutically immunosuppressed to prevent organ rejection. Following resolution of symptomatic dengue, this patient remained positive for DENV3 RNA in the blood for 4months and viruric up to 9months post-infection despite demonstrable levels of serum neutralizing antibodies throughout this period. Full resolution of DENV infection instead coincided with recovery of CD8+ Tcell counts during reversal from lymphopenia. Taken collectively, our observations suggest a role for cellular immunity in sterilizing DENV infection in humans. Any dengue vaccine should thus be able to induce both humoral and cellular immunity that respectively prevent symptomatic infection and enable effective viral clearance.

Enhanced Efficacy of Vaccination With Vaccinia Virus in Old vs. Young Mice.

Immunosenescence is believed to be responsible for poor vaccine efficacy in the elderly. To overcome this difficulty, research into vaccination strategies and the mechanisms of immune responses to vaccination is required. By analyzing the innate and adaptive immune responses to vaccination with vaccinia virus (VACV) in mice of different age groups, we found that immune cell recruitment, production of cytokines/chemokines and control of viral replication at the site of intradermal vaccination were preserved in aged mice and were comparable with younger groups. Analysis of cervical draining lymph nodes (dLN) collected after vaccination showed that numbers of germinal center B cells and follicular T helper cells were similar across different age groups. The number of VACV-specific CD8 T cells in the spleen and the levels of serum neutralizing antibodies 1 month after vaccination were also comparable across all age groups. However, following intranasal challenge of vaccinated mice, body weight loss was lower and virus was cleared more rapidly in aged mice than in younger animals. In conclusion, vaccination with VACV can induce an effective immune response and stronger protection in elderly animals. Thus, the development of recombinant VACV-based vaccines against different infectious diseases should be considered as a strategy for improving vaccine immunogenicity and efficacy in the elderly.

The optimized fusion protein HA1-2-FliCΔD2D3 promotes mixed Th1/Th2 immune responses to influenza H7N9 with low induction of systemic proinflammatory cytokines in mice

H7N9 influenza virus has an unusually high fatality rate of approximately 40%, and a safe and effective vaccine against this subtype is urgently needed. Flagellin, a Toll-like receptor (TLR) 5 agonist, has been deemed as a potent adjuvant candidate. However, its high antigenicity and potential for causing inflammatory injury might restrict its clinical application. Previously, we demonstrated that a fusion protein, HA1-2-FliC, comprising the hemagglutinin globular head protein (HA1-2) of H7N9 influenza virus and the full-length Salmonella typhimurium flagellin protein (FliC), had high efficiency against H7N9 in mouse and chicken models. Here, we constructed an improved fusion protein, HA1-2-FliCΔD2D3, with HA1-2 fused to the FliCΔD2D3 (lacking the hypervariable-region domains D2 and D3 of FliC). HA1-2-FliCΔD2D3 exhibited efficient immunoreactivity and TLR5 agonist efficacy, and promoted innate immune-response activation in mouse macrophages, peripheral blood mononuclear cells, and splenocytes, based on cytokine- and chemokine-expression profiles. Mice immunized with HA1-2-FliCΔD2D3 showed significantly lower systemic inflammatory responses (compared with HA1-2-FliC) and highly reduced flagellin-specific antibody production, without affecting HA1-2-specific antibody production and cellular immune responses. Enhanced IFN-γ/IL-4 generation suggested that HA1-2-FliCΔD2D3 maintained balanced Th1/Th2 immune responses. Furthermore, virus challenge was performed in a chicken model. The results showed that chickens receiving FliCΔD2D3 adjuvant vaccine induced high levels of serum neutralizing antibodies, and exhibited a significant reduction of viral loads in throat and cloaca compared to chickens receiving only HA1-2. In conclusion, we constructed the H7N9 influenza subunit vaccine candidate HA1-2-FliCΔD2D3, with reduced immunogenicity against FliC and lower adverse events. The improved adjuvant FliCΔD2D3 can potentially help in developing safe and effective universal protein-based influenza vaccines for humans.