Maternal immunization with adjuvanted RSV prefusion F protein effectively protects offspring from RSV challenge and alters innate and T cell immunity

Respiratory syncytial virus (RSV) commonly causes severe respiratory tract infections in infants, peaking between 2 and 6 months of age; an age at which direct vaccination is unlikely to be effective. Maternal immunization can deliver high levels of antibodies to newborns, providing immediate protection. Following natural infection, antibodies targeting the prefusion conformation of RSV F protein (PreF) have the greatest neutralizing capacity and thus, may provide infants with a high degree of RSV protection when acquired through maternal vaccination. However, the influence of anti-PreF maternal antibodies on infant immunity following RSV exposure has not been elucidated. To address this knowledge gap, offspring born to dams immunized with a RSV PreF vaccine formulation were challenged with RSV and their immune responses were analyzed over time. These studies demonstrated safety and efficacy for RSV-challenged, maternally-immunized offspring but high and waning maternal antibody levels were associated with differential innate and T cell immunity.     

Maternal immunization with chimpanzee adenovirus expressing RSV fusion protein protects against neonatal RSV pulmonary infection

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract disease with high morbidity and mortality in young infants and children. Despite numerous efforts, a licensed vaccine against RSV remains elusive. Since young infants form the primary target group of RSV disease, maternal immunization to boost the protection in neonates is an attractive strategy. In this study we tested the efficacy of maternal immunization with a chimpanzee adenovirus expressing codon-optimized RSV fusion protein (AdC7-Fsyn) to protect infants against RSV infection. Single intranasal immunization of mice by AdC7-Fsyn induced robust anti-RSV systemic and mucosal immunity that protected against RSV without causing vaccine-enhanced RSV disease. RSV humoral immunity was transferred to pups born to immunized mothers that provided protection against RSV. Immunization with AdC7-Fsyn was effective even in the presence of Ad5 preimmunity. The maternally derived immunity was durable with the half-life of 14.63 days that reduced the viral replication up to 15 weeks of age. Notably, the passively immunized mice could be actively re-immunized with AdC7-Fsyn to boost and extend the protection. This substantiates maternal immunization with an AdC7-based vaccine expressing RSV F as feasible approach to protect against RSV early in life.     

HBHA vaccination may require both Th1 and Th17 immune responses to protect mice against tuberculosis

Almost one century after the discovery of the BCG vaccine, tuberculosis remains a major cause of global mortality and morbidity, emphasizing the urgent need to design more efficient vaccines. The heparin-binding haemagglutinin (HBHA) appears to be a promising vaccine candidate, as it was shown to afford protection to mice against a challenge infection with Mycobacterium tuberculosis when combined with the strong adjuvant DDA/MPL (dimethyldioctadecyl-ammonium bromide/monophosphoryl lipid A), a TLR4 ligand. In this study, we investigated the immunological response and protection of mice immunized with HBHA formulated in lipid-containing nanoparticles and adjuvanted with CpG, a TLR9 ligand. Subcutaneous immunization with this HBHA formulation led to a marked Th1 response, characterized by high IFN-γ levels, but no significant IL-17 production, both in spleen and lung, in contrast to DDA/MPL MPL-formulated HBHA, which induced both IFN-γ and IL-17. This cytokine profile was also observed in BCG-primed mice and persisted after M. tuberculosis infection. No significant protection was obtained against challenge infection after vaccination with the nanoparticle-CpG formulation, and this was associated with a failure to mount a memory immune response. These results suggest the importance of both Th1 and Th17 immune responses for vaccine-induced immunity.     

DDA adjuvant induces a mixed Th1/Th2 immune response when associated with BBG2Na,a respiratory syncytial virus potential vaccine

Human respiratory syncytial virus (hRSV) is one of the most common causes of respiratory infection in infants and the elderly. Previous attempts to vaccinate children against RSV failed and the induction of an aberrant Th2-type immune response was shown to induce severe to fatal pulmonary disease characterised in part by eosinophilia. BBG2Na is a promising human RSV subunit vaccine candidate which successfully passed phase II clinical trials in adults in association with Adju-Phos((R)). However, this formulation is not the most suitable for use in children since aluminium salts are known to induce a Th2-based immune response. In this study, we describe a potent and safe adjuvant formulation for BBG2Na in dimethyldioctadecylammonium bromide (DDA) that induces a mixed Th1/Th2 immune response in BALB/c mice. Furthermore, BBG2Na showed the same protective efficacy against RSV challenge when formulated either in DDA or in alum in mice and cotton rats.     

Immunogenicity and efficacy of recombinant RSV-F vaccine in a mouse model

RSV vaccine development has constraints due to safety issues encountered by formalin-inactivated FI-RSV vaccines. A desirable vaccine should induce Th(1) responses and a strong mucosal immunity to provide complete protection from RSV infection. In the present paper, we developed and evaluated a mucosal vaccine against RSV in a mouse model. The antigenic regions corresponding to residues 412-524 of RSV-F protein were amplified by RT-PCR and cloned into a vector containing the ctxA(2)B gene of the cholera toxin. The recombinant protein was expressed in E. coli and properties of the recombinant protein were analyzed by SDS-PAGE, Western blot and G(M1)-ELISA. The purified recombinant protein (rRF-412) was used to immunize BALB/c mice intranasally. The results from our studies show that the rRF-412 immunogen induced mucosal (IgA) and systemic antibody (IgG, IgG1, IgG2a, and IgG2b) responses which neutralized RSV. The IgG1/IgG2a ratios indicated a Th(1)-biased antibody response. The Th(1) (TNF-alpha, IL-12p70, IFN-gamma, IL-2) and Th(2) (IL-10, IL-4 and IL-5) cytokine profiles were analyzed after stimulation of spleen cells from mice immunized with purified RF-412 protein. Similar to the antibody response, we observed that the rRF-412 immunogen induced a mixed Th(1)/Th(2) cytokine immune response with a Th(1)-bias response. Serum antibodies were capable of neutralizing RSV and mice immunized with rRF-412 were significantly protected from live RSV challenge. Our data provides evidence that the rRF-412 immunogen may be a potential mucosal vaccine candidate against RSV.     

Th17/Th1 biased immunity to the pneumococcal proteins PcsB, StkP and PsaA in adults of different age

Streptococcus pneumoniae is a major human pathogen, causing high morbidity and mortality in children, and also in the elderly, who are particularly susceptible to S. pneumoniae infections due to the dysregulated function of the aged immune system. As the current generation of polysaccharide vaccines do not provide sufficient protection for elderly, new vaccination strategies are urgently needed.To learn whether pneumococcal proteins are able to induce adaptive immune responses in adults in different age groups, we determined serum IgG antibody titers and T cell immunity (IFN-γ, IL-17A and IL-5 production) to three pneumococcal antigens, PcsB, StkP and PsaA, that are components of an investigational protein-based pneumococcal vaccine, IC47. Therefore, sera and PBMCs of 108 healthy adults in three different age groups (young, middle-aged and elderly) were analyzed by ELISA and ELISpot, respectively.We found naturally acquired antibodies to all three proteins in all age groups against all three antigens. However, elderly individuals had significantly lower IgG levels to PcsB and PsaA compared to those of younger donors. There was no significant age-related difference in the overall rate of T cell immunity for the three pneumococcal proteins. We found that the Th17 response was dominant in all age groups and was frequently combined with a Th1 or Th2 response in young and middle-aged subjects. However, in elderly persons there was a lower percentage of PBMC samples producing more than one cytokine upon antigenic stimulation. The narrow cytokine secretion pattern was the most striking difference between elderly and younger adult age groups.Our results demonstrate that in the majority of adults there is a naturally acquired humoral and cellular immune response to the three pneumococcal proteins tested. The dominance of the Th17 response is especially interesting in the light of new insights regarding the role of Th17 cells in mucosal protection against this pathogen.     

Influence of live respiratory syncytial virus priming on the immune response generated by a recombinant vaccine candidate, BBG2Na

Respiratory syncytial virus is one of the major respiratory pathogens for infants and immunocompromized children. With the exception of young children, all the population has encountered RSV and is seropositive. Recent reports have demonstrated however that the virus also affects the elderly and represents a major cause of illness associated with an excess of morbidity and mortality. We have generated a recombinant RSV vaccine, BBG2Na, which is highly protective in rodents against RSV infection. The aim of this study was to evaluate the ability of the vaccine to increase anti-RSV protection in RSV-primed mice and to characterize the induced immune responses.Immunization with BBG2Na increased the anti-RSV-A serum antibody titers of RSV-primed mice with induction of both IgG1 and IgG2a antibodies attesting for a mixed Th response. Moreover, the level of the induced anti-G2Na antibodies was greater in seropositive mice. Finally, sera from RSV-primed mice displayed a higher protective efficacy after transfer into naive mice following subsequent immunization with BBG2Na than sera of mice immunized with RSV-A only.Our results demonstrate that BBG2Na is immunogenic and increases the protective efficacy of serum antibodies in RSV-primed mice; they support the possibility of performing clinical trials in the seropositive human population.     

Both immunisation with a formalin-inactivated respiratory syncytial virus (RSV) vaccine and a mock antigen vaccine induce severe lung pathology and a Th2 cytokine profile in RSV-challenged mice

Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and pneumonia in infants and young children. Immunopathology may play a role in RSV-induced disease and a severe RSV infection may also be associated with an increased risk of developing asthma. Vaccination with formalin-inactivated RSV (FI-RSV) prior to infection resulted both in human and in the mouse model in extensive lung pathology. In the mouse model, it has been shown that this aggravation of disease was associated with a shift in the balance between Th1 and Th2 cytokines towards a Th2-type response. The aim of the present study was to characterise the immunological and inflammatory responses in BALB/c mice upon RSV infection with or without prior vaccination with aluminium-adjuvanted FI-RSV or control antigens (FI-Mock). As previously reported by others, we also observed that a primary RSV infection in BALB/c mice resulted in a predominant Th1-type cytokine response, which was associated with slight bronchiolitis and alveolitis. FI-RSV vaccination prior to RSV challenge prevented virus replication and was associated with an aggravation of pulmonary histopathology and a shift towards a Th2-type response. Vaccination with FI-Mock did not prevent RSV replication in the lung but resulted in an even more pronounced Th2 response after infection while these mice were not sensitised to specific viral antigens. Thus, viral replication in a Th2 responding animal (induced by aluminium-adjuvanted mock vaccine) appears to boost the Th2 response upon RSV infection.     

Long-lasting balanced immunity and protective efficacy against respiratory syncytial virus in mice induced by a recombinant protein G1F/M2

Respiratory syncytial virus (RSV) is the primary cause of serious lower respiratory tract illness in young children. We have engineered a recombinant candidate vaccine G1F/M2, consisting of a cytotoxic T lymphocyte (CTL) epitope of RSV-M2 protein and a domain of RSV-G protein. In this study, the long-term immunogenicity and protective effect were evaluated. In G1F/M2-immunized mice, special antibodies lasted for more than 19 weeks, and the IgG1/IgG2a ratio remained a balanced level till the end of the study, suggesting mixed Th1/Th2 type of responses. Concomitantly, G1F/M2 elicited long-lived RSV-specific CTL activity that was detectable at 12 weeks after the final immunization. Stronger CTL responses were induced with immunization once more at 13 weeks after the last immunization in G1F/M2-primed mice than those in F/M2-primed mice. These results suggest that G1F/M2-induced long-lasting balanced humoral and cellular immunity responses, and immunological memory in mice. Furthermore, following RSV challenge, long-term protective efficacy was observed. RSV replication in lungs of G1F/M2-primed mice elicited also mixed Th1/Th2 responses, a property that is considered advantageous for the safety of an RSV vaccine. Therefore, G1F/M2 is a promising RSV subunit vaccine.     

Prototype Alzheimer's disease epitope vaccine induced strong Th2-type anti-Abeta antibody response with Alum to Quil A adjuvant switch

Beta-amyloid (Abeta) peptide has been proposed to be a causal factor in Alzheimer's disease (AD). Currently being investigated, active and passive Abeta-immunotherapy significantly reduce Abeta plaque deposition, neuritic dystrophy, and astrogliosis in the brains of APP transgenic (APP/Tg) mice. Immunization with Abeta42 formulated in the Th1-type adjuvant QS21 was beneficial for AD patients with significant titers of anti-Abeta antibodies, however, 6% of participants developed meningoencephalitis, likely due to anti-Abeta-specific autoimmune Th1 cells. Thus, successful Abeta vaccination requires the development of strong antibody responses without Th1-type cellular immunity. In this study, we compared the induction of humoral immune responses with Th1-type (Quil A) and Th2-type (Alum) adjuvants singly and in combination, using our novel epitope vaccine composed of self B cell epitope Abeta(1-15) and foreign T cell epitope PADRE (PADRE-Abeta(1-15)-MAP). Formulated in Quil A, this vaccine resulted in significantly higher anti-Abeta antibody responses in both BALB/c (H-2d) and C57BL/6 (H-2b) mice, compared with Alum. Anti-Abeta antibodies induced by Alum were predominantly IgG1 type accompanied by lower levels of IgG2a and IgG2b. Quil A induced robust and almost equal titers of anti-Abeta antibodies of IgG1 and IgG2a isotypes and slightly lower levels of IgG2b. Switching adjuvants from Alum to Quil A induced higher concentrations of antibodies than injections with Alum only, however slightly lower than Quil A only. Switching both adjuvants did not change the profile of antibody responses generated by the initial adjuvant injected. These results suggest that switching from Alum to Quil A would be beneficial for AD patients because anti-Abeta antibody production was enhanced without changing the initially generated and likely beneficial Th2-type humoral response.     

Intraperitoneal immunization of recombinant HSP70 (DnaK) of Salmonella Typhi induces a predominant Th2 response and protective immunity in mice against lethal Salmonella infection

Heat shock proteins serve as important antigens in defense against infectious diseases. Members of HSP70 family, particularly microbial HSP70s have acquired special significance in immunity. In the present study, we evaluated the immunogenicity and protective efficacy of HSP70 of Salmonella enterica serovar Typhi against lethal infection by Salmonella in mice with or without adjuvants. The HSP70 gene was cloned and expressed in Escherichia coli BL21 and purified by affinity chromatography. Immunization of mice with HSP70 either alone or in combination with complete Freund's adjuvant (CFA) resulted in a significant increase in antibody titers as compared to control. Antibody isotyping revealed that HSP70 immunization induces both IgG1 and IgG2a antibodies to a significant extent but a higher IgG1/IgG2a ratio indicates a predominant Th2 response. There was a significant increase in lymphocyte proliferation, and levels of both Th2 and Th1 cytokines in cells isolated from immunized mice as compared to control. Immunization of mice with recombinant HSP70 either alone or in combination with CFA conferred 70-90% protection against lethal infections by Salmonella Typhi Ty2 or Salmonella Typhimurium. However, passive immunization with anti-HSP70 sera induced only partial protection in the immunized mice.     

A promising balanced Th1 and Th2 directing immunological adjuvant, saponins from the root of Platycodon grandiflorum

The haemolytic activities and adjuvant potentials of Platycodon grandiflorum saponin (PGS) and its fractions on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA) were evaluated. PGS was subjected to silica gel column chromatography to afford four fractions, and two fractions PGSC and PGSD selected for testing for activities because of containing dominant saponin peaks. PGS, PGSC, and PGSD showed a slight haemolytic effect, with their HD50 value being 37.91+/-2.24, 21.30+/-1.22, 37.58+/-1.86 microg/ml against 0.5% rabbit red blood cell, respectively. ICR mice were immunized subcutaneously with OVA 100 microg alone or with OVA 100 microg dissolved in saline containing Alum (200 microg), Quil A (10 microg), PGS (50, 100 or 200 microg), PGSC, or PGSD (25, 50 or 100 microg) on days 1 and 15. Two weeks later (day 28), concanavalin A (Con A)-, pokeweed (PWM)-, and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. PGS and PGSC significantly enhanced the Con A-, PWM-, and OVA-induced splenocyte proliferation in OVA-immunized mice at three doses (P<0.01 or P<0.001). However, no significant differences (P>0.05) were observed among the OVA group, OVA/Alum group and OVA/PGSD group. OVA-specific IgG, IgG1, and IgG2b antibody levels in serum were significantly enhanced by PGS, PGSC, and PGSD compared with OVA control group (P<0.05, P<0.01, or P<0.001). Moreover, the adjuvant effects of PGSC (50 or 100 microg) on the OVA-specific IgG, IgG1, and IgG2b antibody responses to OVA in mice were more significant than those of Alum. In conclusion, PGS seem to be a promising balanced Th1 and Th2 directing immunological adjuvants which can enhance the immunogenicity of vaccine.     

Recombinant SARS-CoV-2 RBD with a built in T helper epitope induces strong neutralization antibody response

Without approved vaccines and specific treatments, COVID-19 is spreading around the world with above 26 million cases and approximately 864 thousand deaths until now. An efficacious and affordable vaccine is urgently needed. The Val308 – Gly548 of spike protein of SARS-CoV-2 linked with Gln830 – Glu843 of Tetanus toxoid (TT peptide) (designated as S1-4) and without TT peptide (designated as S1-5) were expressed and renatured. The antigenicity and immunogenicity of S1-4 were evaluated by Western Blotting (WB) in vitro and immune responses in mice, respectively. The protective efficiency was measured preliminarily by microneutralization assay (MN50). The soluble S1-4 and S1-5 protein was prepared to high homogeneity and purity. Adjuvanted with Alum, S1-4 protein stimulated a strong antibody response in immunized mice and caused a major Th2-type cellular immunity supplemented with Th1-type immunity. Furthermore, the immunized sera could protect the Vero E6 cells from SARS-CoV-2 infection with neutralizing antibody titer 256. Recombinant SARS-CoV-2 RBD with a built in T helper epitope could stimulate both strong humoral immunity supplemented with cellular immunity in mice, demonstrating that it could be a promising subunit vaccine candidate.     

Baculovirus-expressed virus-like particle vaccine in combination with DNA encoding the fusion protein confers protection against respiratory syncytial virus

Respiratory syncytial virus (RSV) is a major viral agent causing significant morbidity and mortality in young infants and the elderly. There is no licensed vaccine against RSV and it is a high priority to develop a safe RSV vaccine. We determined the immunogenicity and protective efficacy of combined virus-like particle and DNA vaccines presenting RSV glycoproteins (Fd.VLP) in comparison with formalin inactivated RSV (FI-RSV). Immunization of mice with Fd.VLP induced higher ratios of IgG2a/IgG1 antibody responses compared to those with FI-RSV. Upon live RSV challenge, Fd.VLP and FI-RSV vaccines were similarly effective in clearing lung viral loads. However, FI-RSV immunized mice showed a substantial weight loss and high levels of T helper type 2 (Th2) cytokines as well as extensive lung histopathology and eosinophil infiltration. In contrast, Fd.VLP immunized mice did not exhibit Th2 type cytokines locally and systemically, which might contribute to preventing vaccine-associated RSV lung disease. These results indicate that virus-like particles in combination with DNA vaccines represent a potential approach for developing a safe and effective RSV vaccine.     

A M2 protein-based universal influenza vaccine containing Advax-SM adjuvant provides newborn protection via maternal or neonatal immunization

BackgroundDespite newborns being at increased risk of serious influenza infection, influenza vaccines are currently not recommended for use in infants under 6 months of age. We therefore sought to evaluate the protective efficacy in mice of an M2-based influenza vaccine (CapM2e) formulated with Advax-SM adjuvant. Vaccine protection was assessed via both passive maternal immunization and direct neonatal immunization.MethodsFor maternal transfer studies, female mice were immunized 1 week before and after mating. Blood was collected from both mother and offspring during weaning and pups were challenged when they reached 3 weeks of age with lethal doses of H1N1 and homologous reassortment influenza strain H3N2 with conserved M2. For direct immunization studies, newborns were immunized at 1 and 3 weeks of age and blood was collected prior to challenge at 4 weeks of age.ResultsMaternal immunization with CapM2e + Advax-SM vaccine induced high maternal M2e antibody levels that were passively transferred to their offspring and provided them with protection against both H1N1 and H3N2 influenza strains when challenged at 3 weeks of age. When used for direct immunization of neonatal mice, CapM2e + Advax-SM vaccine similarly induced high serum M2e antibody levels and protected against H1N1 and H3N2 influenza challenges with protection associated with inhibition of virus replication with a significant reduction in lung virus load in immunized pups.ConclusionCapM2e + Advax-SM vaccine could be useful for protecting newborns against diverse influenza A strains, with opportunities to achieve protection by passive maternal immunization or active neonatal immunization. This data supports further development of this promising M2e-based vaccine candidate.     

Th17/IL-17与呼吸道合胞病毒毛细支气管炎气道炎症相关性探讨

目的 通过检测呼吸道合胞病毒(respiratory syncytial virus, RSV)毛细支气管炎(毛支)患儿外周血中Th17细胞及细胞因子IL-17的水平变化, 探讨其与RSV毛支气道炎症的相关性。方法 选择40例初次喘息发作的RSV毛支患儿作为研究对象, 急性期及恢复期各抽取外周静脉血2 mL, 流式细胞术检测Th-17细胞比例, ELISA法检测IL-17水平。结果 RSV毛支急性期Th17及IL-17水平均明显升高(P＜0.01);与恢复期相比, 差异有统计学意义;重度毛支与轻度毛支相比, 急性期Th17细胞比例及IL-17水平明显升高(P＜0.01), 而恢复期差异无统计学意义(P＞0.05)。结论 Th17及IL-17可能参与RSV毛支急性期气道炎症反应过程, 并与疾病的严重程度相关。     

Poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) is a potent enhancer of mixed Th1/Th2 immune responses in mice immunized with influenza virus antigens

We investigated the ability of a novel polyphosphazene polyelectrolyte, poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) to enhance antigen-specific immune responses. BALB/c mice were immunized once subcutaneously with either bovine serum albumin (BSA) or influenza virus X:31 antigen alone, or in combination with PCEP, or either of the adjuvants poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP) and alum. Both PCEP and PCPP significantly enhanced serum antigen-specific total IgG, IgG1 and IgG2a antibody titers, and these responses were highest in PCEP-immunized mice. Alum induced only a modest enhancement of antibody responses. Reducing the dose of X:31 antigen by 25-fold had no effect on antibody responses in mice immunized with PCPP and PCEP, but resulted in reduced titers in those immunized with alum. Analysis of X:31 antigen-specific cytokines revealed that alum and PCPP were associated with a predominantly IL-4 response. In contrast, PCEP was associated with production of both IFNgamma and IL-4. We conclude that PCEP is a potent enhancer of antigen-specific Th1 and Th2 immune responses and is a promising adjuvant for vaccine applications.     

A novel adjuvant G3 induces both Th1 and Th2 related immune responses in mice after immunization with a trivalent inactivated split-virion influenza vaccine

A preferred adjuvant should promote both Th1 and Th2 responses. However, most adjuvants in common use are biased towards a Th2-driven response. Therefore, the ability of a novel saponin-based adjuvant G3 to inducing balanced Th1 and Th2 responses in BALB/c mice immunized with a split trivalent seasonal influenza vaccine was evaluated in comparison to that of the adjuvant Al(OH)₃. Clear differences in the IgG profiles induced by G3, Al(OH)₃ or non-adjuvanted vaccine were recorded. Both adjuvants enhanced high and similar levels of the Th2 associated IgG1 subtype compared to mice given vaccine alone. Only G3 enhanced the IgG2a subclass reflecting a Th1 response, whereas Al(OH)₃ even abrogated the IgG2a production. Accordingly, G3 enhanced the production of IL-2 and IFN-γ and also of IL-2/IFN-γ double secreting cells, emphasizing the strong Th1 driving effect of G3. Only Al(OH)₃ increased splenocyte production of IL-17. Taken together, the results indicate a strong propensity for G3 to induce both Th1 and Th2 driven immune responses.