Maternal immunization with respiratory syncytial virus fusion protein formulated with a novel combination adjuvant provides protection from RSV in newborn lambs

Respiratory syncytial virus (RSV) is the causative agent of serious upper and lower respiratory tract infections in newborns and infants. Protection from RSV is crucial for neonates, and maternal immunization is one approach that holds promise for providing immediate protection to young infants against severe RSV infection. We previously reported efficacy of a subunit vaccine consisting of the fusion (F) protein formulated with a novel adjuvant (ΔF/TriAdj) in neonates. The goal of the current study was to evaluate the ΔF/TriAdj as a maternal vaccine. Pregnant ewes were vaccinated intramuscularly with ΔF/TriAdj or PBS six weeks prior to lambing, and re-vaccinated four weeks later, which resulted in transfer of maternal antibodies (MatAbs) to the newborn lambs through the colostrum. Significantly higher levels of RSV ΔF-specific serum IgG were detected in vaccinated pregnant ewes and their lambs when compared to control animals, which revealed that MatAbs were passively transferred to the offspring. All newborn lambs were challenged with RSV at three days of age. After RSV challenge, virus production and lung pathology were significantly lower in lambs that had received passively transferred antibodies than in control animals. These results indicate that maternal immunization with ΔF/TriAdj might be an alternative, safe and effective approach to provide protection against RSV in newborn and young infants.     

Maternal immunization with RSV fusion glycoprotein vaccine and substantial protection of neonatal baboons against respiratory syncytial virus pulmonary challenge

Globally, human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory infection in infants and young children. There are no licensed vaccines despite the high worldwide disease burden. RSV fusion (F) glycoprotein vaccine is the most advanced candidate for maternal immunization. In this report, a baboon maternal immunization model was used to assess the immunogenicity and protection of infants against pulmonary challenge with human RSV/A. Vaccination in the third trimester produced high anti-RSV F IgG titers and virus-neutralizing antibodies. Infants born to immunized females had high levels of serum RSV antibodies that were comparable to maternal levels at birth and persisted for over 50 days with a half-life of 14–24 days. Furthermore, infants from immunized females and challenged with RSV/A were healthy, developed less severe disease, and had only mild pulmonary inflammatory changes whereas infants born to non-vaccinated females developed more severe disease with marked to moderate interstitial pneumonia, pulmonary edema, and bronchiolar obstruction. These results support the further development of the RSV F vaccine for maternal immunization.     

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.     

Formulation of the prefusion RSV F protein with a Th1/Th2-balanced adjuvant provides complete protection without Th2-skewed immunity in RSV-experienced young mice

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections among infants with most infections occurring in the first year of life. Multiple RSV exposures are required for children to mount adult-like immune responses. Although adult RSV immunity is associated with less severe disease, the protection induced through natural infection is short-lived. Therefore, vaccination of RSV-experienced young children may accelerate immunity and provide long-term protection from RSV reinfection. However, the extent to which different Th-biased vaccine regimens influence pre-existing humoral and cellular immunity in RSV-experienced young children is unknown. To address this question, infant BALB/c mice were RSV-infected and subsequently immunized with the prefusion RSV F (PreF) antigen formulated with either a Th2-skewing (Alum) or Th1/Th2-balanced (Advax-SM) adjuvant. These studies show that both adjuvants boosted neutralizing antibody and protected from RSV reinfection, but Advax-SM adjuvant prevented the Th2-skewed immunity observed in RSV-experienced young mice immunized with PreF/Alum.     

An insect cell derived respiratory syncytial virus (RSV) F nanoparticle vaccine induces antigenic site II antibodies and protects against RSV challenge in cotton rats by active and passive immunization

Post-infectious immunity to respiratory syncytial virus (RSV) infection results in limited protection as evidenced by the high rate of infant hospitalization in the face of high titer, maternally derived RSV-specific antibodies. By contrast, RSV fusion (F) glycoprotein antigenic site II humanized monoclonal antibodies, palivizumab and motavizumab, have been shown to reduce RSV-related hospitalization in infants. Immunogenicity and efficacy studies were conducted in cotton rats comparing a recombinant RSV F nanoparticle vaccine with palivizumab and controlled with live RSV virus intranasal immunization and, formalin inactivated RSV vaccine. Active immunization with RSV F nanoparticle vaccine containing an alum adjuvant induced serum levels of palivizumab competing antibody (PCA) greater than passive administration of 15 mg/kg palivizumab (human prophylactic dose) in cotton rats and neutralized RSV-A and RSV-B viruses. Immunization prevented detectable RSV replication in the lungs and, unlike passive administration of palivizumab, in the nasal passage of challenged cotton rats. Histology of lung tissues following RSV challenge showed no enhanced disease in the vaccinated groups in contrast to formalin inactivated ‘Lot 100’ vaccine. Passive intramuscular administration of RSV F vaccine-induced immune sera one day prior to challenge of cotton rats reduced viral titers by 2 or more log₁₀ virus per gram of lung and nasal tissue and at doses less than palivizumab. A recombinant RSV F nanoparticle vaccine protected lower and upper respiratory tract against both RSV A and B strain infection and induced polyclonal palivizumab competing antibodies similar to but potentially more broadly protective against RSV than palivizumab.     

Intranasal immunization with HRSV prefusion F protein and CpG adjuvant elicits robust protective effects in mice

Human respiratory syncytial virus (HRSV) is a leading cause of lower respiratory tract infections in elderly individuals and young children/infants and can cause bronchiolitis and even death. There is no licensed HRSV vaccine. An ideal vaccine should induce high titers of neutralizing antibodies and a Th1-biased immune response. In this study, we used EXPI293 cells to express the fusion (F) protein with a prefusion conformation (PrF) and compared the safety and efficacy of intranasal immunization with PrF in combination with two mucosal adjuvants (CpG ODN and liposomes) in mice. After two intranasal administrations, mice in the PrF + CpG group produced high titers of neutralizing antibodies (4961) and a Th1-biased immune response compared with the PrF + Lipo group. The lung viral load of mice in the PrF + CpG group was significantly reduced (3.5 log) compared with that in the adjuvant control group, and the survival rate was 100 %, while the survival rate of mice in the PrF + Lipo group was only 67 %. At the same time, this immunization strategy reduced the pathological damage to the lungs in mice. In conclusion, the combination of PrF and CpG adjuvant is immunogenic, elicits a Th1 type immune response, and completely protects mice from a lethal HRSV challenge. It is worthy of further evaluation as an HRSV vaccine in clinical trials.Clinical trial registration.This study was not related to human participation or experimentation.     

Immunogenicity and protective efficacy of adenoviral and subunit RSV vaccines based on stabilized prefusion F protein in pre-clinical models

Respiratory Syncytial Virus (RSV) remains a leading cause of severe respiratory disease for which no licensed vaccine is available. We have previously described the derivation of an RSV Fusion protein (F) stabilized in its prefusion conformation (preF) as vaccine immunogen and demonstrated superior immunogenicity in naive mice of preF versus wild type RSV F protein, both as protein and when expressed from an Ad26 vaccine vector. Here we address the question if there are qualitative differences between the two vaccine platforms for induction of protective immunity. In naïve mice, both Ad26.RSV.preF and preF protein induced humoral responses, whereas cellular responses were only elicited by Ad26.RSV.preF. In RSV pre-exposed mice, a single dose of either vaccine induced cellular responses and strong humoral responses. Ad26-induced RSV-specific cellular immune responses were detected systemically and locally in the lungs. Both vaccines showed protective efficacy in the cotton rat model, but Ad26.RSV.preF conferred protection at lower virus neutralizing titers in comparison to RSV preF protein. Factors that may contribute to the protective capacity of Ad26.RSV.preF elicited immunity are the induced IgG2a antibodies that are able to engage Fcγ receptors mediating Antibody Dependent Cellular Cytotoxicity (ADCC), and the induction of systemic and lung resident RSV specific CD8 + T cells. These data demonstrate qualitative improvement of immune responses elicited by an adenoviral vector based vaccine encoding the RSV preF antigen compared to the subunit vaccine in small animal models which may inform RSV vaccine development.     

Local innate and adaptive immune responses regulate inflammatory cell influx into the lungs after vaccination with formalin inactivated RSV

Inactivated respiratory syncytial virus (RSV) vaccines tend to predispose for immune mediated enhanced disease, characterized by Th2 responses and airway hypersensitivity reactions. We show in a C57BL/6 mouse model that the early innate response elicited by the challenge virus (RSV versus influenza virus) influences the outcome of the Th1/Th2 balance in the lung after intramuscular priming with inactivated vaccine. Priming of CD4⁺/IFN-γ⁺ T cells by mature dendritic cells administered intravenously and/or priming of a virus specific CD8⁺ T cell response ameliorated the Th2-mediated inflammatory response in the lung, suggesting that vaccination procedures are feasible that prevent vaccine induced immune pathology.     

Formulation of the respiratory syncytial virus fusion protein with a polymer-based combination adjuvant promotes transient and local innate immune responses and leads to improved adaptive immunity

Respiratory syncytial virus (RSV) causes serious upper and lower respiratory tract infections in newborns and infants. Presently, there is no licensed vaccine against RSV. We previously reported the safety and efficacy of a novel vaccine candidate (ΔF/TriAdj) in rodent and lamb models following intranasal immunization. However, the effects of the vaccine on the innate immune system in the upper and lower respiratory tracts, when delivered intranasally, have not been characterized. In the present study, we found that ΔF/TriAdj triggered transient production of chemokines, cytokines and interferons in the nasal tissues and lungs of BALB/c mice. The types of chemokines produced were consistent with the populations of immune cells recruited, i.e. dendritic cells, macrophages and neutrophils, in the nose-associated lymphoid tissue (NALT), lung and their draining lymph nodes of the ΔF/TriAdj-immunized group. In addition, ΔF/TriAdj stimulated cellular activation with generation of mucosal and systemic antibody responses, and conferred complete protection from viral infection in the lungs upon RSV challenge. The effect of ΔF/TriAdj was short-lived in the nasal tissues and more prolonged in the lungs. In addition, both innate and adaptive immune responses were lower when mice were immunized with ΔF alone. These results suggest that ΔF/TriAdj modulates the innate mucosal environment in both upper and lower respiratory tracts, which contributes to robust adaptive immune responses and long-term protective efficacy of this novel vaccine formulation.     

Local innate responses and protective immunity after intradermal immunization with bovine viral diarrhea virus E2 protein formulated with a combination adjuvant in cattle

Bovine viral diarrhea virus (BVDV) is one of the most serious pathogens in cattle. Recently, we developed a novel adjuvant platform (TriAdj) that includes a toll-like receptor 3 agonist, poly (I:C); an innate defense regulatory peptide; and water-soluble polymer, poly[di(sodiumcarboxylatoethylphenoxy)]-phosphazene (PCEP). To develop a needle-free intradermal (ID) subunit vaccine, the BVDV type-2 E2 protein was formulated with TriAdj, and immune protection was evaluated in calves against a BVDV-2 strain. Intradermal delivery of E2/TriAdj elicited robust virus neutralizing antibodies and cell-mediated immune responses including CD4⁺ and CD8⁺ T-cell responses. The development of CD8⁺ T-cell responses in vaccinated calves indicates that TriAdj promotes cross-presentation. Upon challenge with virulent BVDV-2, the vaccinated calves showed no weight loss, leukopenia or virus shedding, and almost no temperature increase, in contrast to the control animals, which had severe clinical disease and shed virus for three to six days in nasal fluids and white blood cells. Intradermal vaccination was shown to attract various immune cell populations including dendritic cells, the most important antigen presenting cells. These data demonstrate that ID delivery is suitable as an administration route in cattle and that ID delivered, TriAdj-formulated E2 can protect cattle from BVDV-2.     

A rabies vaccine adjuvanted with saponins from leaves of the soap tree (Quillaja brasiliensis) induces specific immune responses and protects against lethal challenge

Quillaja brasiliensis (Quillajaceae) is a saponin producing species native from southern Brazil and Uruguay. Its saponins are remarkably similar to those of Q. saponaria, which provides most of the saponins used as immunoadjuvants in vaccines. The immunostimulating capacities of aqueous extract (AE) and purified saponin fraction (QB-90) obtained from leaves of Q. brasiliensis were favorably comparable to those of a commercial saponin-based adjuvant preparation (Quil-A^®) in experimental vaccines against bovine herpesvirus type 1 and 5, poliovirus and bovine viral diarrhea virus in mice model. Herein, the immunogenicity and protection efficacy of rabies vaccines adjuvanted with Q. brasiliensis AE and its saponin fractions were compared with vaccines adjuvanted with either commercial Quil-A or Alum. Mice were vaccinated with one or two doses (on days 0 and 14) of one of the different vaccines and serum levels of total IgG, IgG1 and IgG2a were quantified over time. A challenge experiment with a lethal dose of rabies virus was carried out with the formulations. Viral RNA detection in the brain of mice was performed by qPCR, and RNA copy-numbers were quantified using a standard curve of in vitro transcribed RNA. All Q. brasiliensis saponin-adjuvanted vaccines significantly enhanced levels of specific IgG isotypes when compared with the no adjuvant group (P ≤ 0.05). Overall, one or two doses of saponin-based vaccine were efficient to protect against the lethal rabies exposure. Both AE and saponin fractions from Q. brasiliensis leaves proved potent immunological adjuvants in vaccines against a lethal challenge with a major livestock pathogen, hence confirming their value as competitive or complementary sustainable alternatives to saponins of Q. saponaria.     

Mucosal immunization with experimental feline immunodeficiency virus (FIV) vaccines induces both antibody and T cell responses but does not protect against rectal FIV challenge

Feline immunodeficiency virus (FIV) is a natural lentiviral pathogen of cats which can be experimentally transmitted via rectal and vaginal routes — the major routes of human immunodeficiency virus type 1 transmission in man. An important objective for lentiviral research is the development of vaccine strategies which generate good mucosal immune responses capable of giving protection from a mucosal virus challenge. The experimental vaccines employed in this study were based on (a) a peptide from the third variable region of the FIV envelope glycoprotein and (b) fixed whole FIV, Glasgow-8 strain. Adjuvants used were Quil A and cholera toxin for mucosal administration and incomplete Freund’s adjuvant and immune stimulating complexes for subcutaneous injection. Mucosal immunization was given by rectal and intranasal routes. Both antibody and proliferative responses were elicited by mucosal immunization and cholera toxin was found to be a good mucosal adjuvant. The addition of a lipo thioester to the FIV peptide improved IgG and IgA responses upon parenteral administration. However, no protection from a rectal FIV challenge was achieved.     

Recombinant Erns-E2 protein vaccine formulated with MF59 and CPG-ODN promotes T cell immunity against bovine viral diarrhea virus infection

To obtain an effective vaccine candidate against bovine viral diarrhea virus (BVDV) disease which causes great economical loss in cattle industries, recombinant E^rns-E2 protein vaccine containing MF59 and CPG-ODN adjuvants was prepared and assessed in this study. The recombinant plasmid (pET32a-E^rns-E2) was constructed and transformed into BL21 (DE3) cells to produce E^rns-E2 protein. We immunized mice with the MF59–and CPG-ODN–adjuvanted recombinant E^rns-E2 protein, E2 protein, or E^rns protein, respectively. To evaluate immunogenicity and efficacy of a vaccine-adjuvant combination, mice were challenged with BVDV BJ175170 strain after immunization. All adjuvanted vaccines elicited detectable humoral and cellular immune responses, the BVDV-specific antibody titers as well as interleukin 4 (IL-4) levels in sera of mice immunized with the recombinant E^rns-E2 protein were higher than in those of mice immunized with either the recombinant E^rns or E2 protein. Besides, immunization with the E^rns-E2 vaccines induced higher percentage of CD4⁺IFN-γ⁺, CD8⁺IFN-γ⁺ T cells and CD3⁺TNF-α⁺ T cells compared with the other vaccines. More protective efficacy against BVDV infection was acquired in the mice treated with the recombinant E^rns-E2 protein, as shown by a reduction of viremia and slight pathological changes compared with both the control mice and the other vaccinated mice. Our findings suggest that the use of the recombinant E^rns-E2 protein vaccine formulated with MF59 and CPG-ODN adjuvants enhances T cell responses and viral control, which warrants the E^rns-E2 protein vaccine-adjuvant combination could be as a vaccine strategy to against BVDV.     

Immunization with BLS-Stx2B chimera totally protects dams from early pregnancy loss induced by Shiga toxin type 2 (Stx2) and confers anti-Stx2 immunity to the offspring

Shiga toxin producing Escherichia coli (STEC) are bacterial pathogens involved in food-borne diseases. Shiga toxin (Stx) is the main virulence factor of STEC and is responsible for systemic complications including Hemolytic Uremic Syndrome (HUS). It has been previously demonstrated that Shiga toxin type 2 (Stx2) induces pregnancy loss in rats in early stage of pregnancy. The main purpose of this study was to determine if an active immunization prevents Stx2 mediated pregnancy loss and confers passive protective immunity to the offspring. For that purpose Sprague Dawley female rats were immunized with the chimera based on the enzyme lumazine synthase from Brucella spp. (BLS) and the B subunit of Shiga toxin 2 (Stx2B) named BLS-Stx2B. After immunization females were mated with males. At day 8 of gestation, dams were challenged intraperitoneally with a sublethal and abortifacient dose of Stx2. The immunization induced high anti-Stx2B-specific antibody titers in sera and most important, prevented pregnancy loss. Pups born and breastfeed by immunized dams had high anti-Stx2B-specific antibody titers in sera. Cross-fostering experiments indicated that passive protective immunity against Stx2 was transmitted through lactation. These results indicate that immunization of adult female rats with BLS-Stx2B prevents Stx2-induced pregnancy loss and confers anti Stx2 protective immunity to the offspring.     

Nasal immunization of mice with AFCo1 or AFPL1 plus capsular polysaccharide Vi from Salmonella typhi induces cellular response and memory B and T cell responses

The response to infection against Salmonella involves both B and T cell mediated immunity. An effective immunization can activate an adequate immune response capable to control the primary infection and protect against a secondary infection. Mucosal vaccination, by inducing local pathogen-specific immune responses, has the potential to counter mucosally transmitted pathogens at the portal of entry, thereby increasing the efficacy of vaccines. The aim of this work was to explore the efficacy of AFCo1 or AFPL1, as mucosal adjuvants to stimulate cell immunity and memory responses against Vi polysaccharide antigen of Salmonella typhi (PsVi). Mice immunized with 3 intranasal doses exhibited high levels of PsVi-specific IgG (p < 0.05), IgG2a and IgG2c subclasses. Also, an amplified recall response after a booster immunization with a plain polysaccharide vaccine was induced. Avidities index were higher in mice immunized with adjuvanted formulations at different chaotropic concentrations. Furthermore, IL-12 and IFN-γ levels in nasally vaccinated mice with both adjuvants were induced. Moreover, priming with 3 doses followed by booster immunization with VaxTyVi^® resulted in high levels of anti-Vi specific IgG, IgG subclasses and antibody avidity. Long lived plasma cells in bone marrow, memory B cells and long-term memory T cells after booster dose were induced. The combined formulation of Vi polysaccharide with mucosal adjuvants provides an improved immunogenicity, in particular with regard to cellular responses and long lasting cells responses.     

A cell culture-derived whole-virus H9N2 vaccine induces high titer antibodies against hemagglutinin and neuraminidase and protects mice from severe lung pathology and weight loss after challenge with a highly virulent H9N2 isolate

Background

Influenza viruses of subtype A/H9N2 are enzootic in poultry across Asia and the Middle East and are considered to have pandemic potential. The development of new vaccine manufacturing technologies is a cornerstone of influenza pandemic preparedness.

Methods

A non-adjuvanted whole-virus H9N2 vaccine was developed using Vero cell culture manufacturing technology. The induction of hemagglutination inhibition (HI) and virus-neutralizing antibodies was assessed in CD1 mice and guinea pigs. A highly sensitive enzyme-linked lectin assay was used to investigate the induction of antibodies capable of inhibiting the enzymatic activity of the H9N2 neuraminidase. Protective efficacy against virus replication in the lung after challenge with the homologous virus was evaluated in BALB/c mice by a TCID₅₀ assay, and prevention of virus replication in the lung and associated pathology were evaluated by histology and immunohistochemistry. To investigate the ability of the vaccine to prevent severe disease, BALB/c mice were challenged with a highly virulent mouse-adapted H9N2 isolate which was generated by multiple lung-to-lung passage of wild-type virus.

Results

The vaccine elicited high titers of functional H9N2-specific HA antibodies in both mice and guinea pigs, as determined by HI and virus neutralization assays. High titer H9N2-specific neuraminidase inhibiting (NAi) antibodies were also induced in both species. Vaccinated mice were protected from lung virus replication in a dose-dependent manner after challenge with the homologous H9N2 virus. Immunohistochemical analyses confirmed the lack of virus replication in the lung and an associated substantial reduction in lung pathology. Dose-dependent protection from severe weight loss was also provided after challenge with the highly virulent mouse-adapted H9N2 virus.

Conclusions

The induction of high titers of H9N2-specific HI, virus-neutralizing and NAi antibodies and dose-dependent protection from virus replication and severe disease in animal models suggest that the Vero cell culture-derived whole-virus vaccine will provide an effective intervention in the event of a H9N2 pandemic situation.