Ebola virus glycoprotein Fc fusion protein confers protection against lethal challenge in vaccinated mice

Ebola virus is a Filoviridae that causes hemorrhagic fever in humans and induces high morbidity and mortality rates. Filoviruses are classified as “Category A bioterrorism agents”, and currently there are no licensed therapeutics or vaccines to treat and prevent infection. The Filovirus glycoprotein (GP) is sufficient to protect individuals against infection, and several vaccines based on GP are under development including recombinant adenovirus, parainfluenza virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus (VSV) and virus-like particles. Here we describe the development of a GP Fc fusion protein as a vaccine candidate. We expressed the extracellular domain of the Zaire Ebola virus (ZEBOV) GP fused to the Fc fragment of human IgG1 (ZEBOVGP-Fc) in mammalian cells and showed that GP undergoes the complex furin cleavage and processing observed in the native membrane-bound GP. Mice immunized with ZEBOVGP-Fc developed T-cell immunity against ZEBOV GP and neutralizing antibodies against replication-competent VSV-G deleted recombinant VSV containing ZEBOV GP. The ZEBOVGP-Fc vaccinated mice were protected against challenge with a lethal dose of ZEBOV. These results show that vaccination with the ZEBOVGP-Fc fusion protein alone without the need of a viral vector or assembly into virus-like particles is sufficient to induce protective immunity against ZEBOV in mice. Our data suggested that Filovirus GP Fc fusion proteins could be developed as a simple, safe, efficacious, and cost effective vaccine against Filovirus infection for human use.     

Marburg virus-like particles protect guinea pigs from lethal Marburg virus infection

Ongoing outbreaks of filoviruses in Africa and concerns about their use in bioterrorism attacks have led to intense efforts to find safe and effective vaccines to prevent the high mortality associated with these viruses. We previously reported the generation of virus-like particles (VLPs) for the filoviruses, Marburg (MARV) and Ebola (EBOV) virus, and that vaccinating mice with Ebola VLPs (eVLPs) results in complete survival from a lethal EBOV challenge. The objective of this study was to determine the efficacy of Marburg VLPs (mVLPs) as a potential vaccine against lethal MARV infection in a guinea pig model. Guinea pigs vaccinated with mVLPs or inactivated MARV developed MARV-specific antibody titers, as tested by ELISA or plaque-reduction and neutralization assays and were completely protected from a MARV challenge over 2000 LD50. While eVLP vaccination induced high EBOV-specific antibody responses, it did not cross-protect against MARV challenge in guinea pigs. Vaccination with mVLP or eVLP induced proliferative responses in vitro only upon re-exposure to the homologous antigen and this recall proliferative response was dependent on the presence of CD4+ T cells. Taken together with our previous work, these findings suggest that VLPs are a promising vaccine candidate for the deadly filovirus infections.     

SIV vaccines: current status. The role of the SIV-macaque model in AIDS research.

SIV vaccines made of inactivated whole virus, modified live virus and native and recombinant envelope antigens have protected macaques against experimental infection with low doses of cell-free SIV given systemically. The few vaccinated monkeys that do become infected have tended to live longer than the infected controls. Protection against cell-associated virus has not as yet been tested. The recombinant envelope vaccines now on test have generally not been as effective as the whole virus vaccines. Post-infectious immunotherapy with SIV vaccines has been ineffective. The same whole virus and modified live virus vaccines that protect against systemic infection fail to protect against genital mucosal challenge with cell-free virus. Since sexual transmission is the major route of HIV spread on a global scale, a major effort is now required to develop vaccines in this animal model that induce genital mucosal as well as systemic immunity against infection with both cell-free and cell-associated SIV.     

Virus-like particles exhibit potential as a pan-filovirus vaccine for both Ebola and Marburg viral infections

A safe and effective pan-filovirus vaccine is highly desirable since the filoviruses Ebola virus (EBOV) and Marburg virus (MARV) cause highly lethal disease typified by unimpeded viral replication and severe hemorrhagic fever. Previously, we showed that expression of the homologous glycoprotein (GP) and matrix protein VP40 from a single filovirus, either EBOV or MARV, resulted in formation of wild-type virus-like particles (VLPs) in mammalian cells. When used as a vaccine, the wild-type VLPs protected from homologous filovirus challenge. The aim of this work was to generate a multi-agent vaccine that would simultaneously protect against multiple and diverse members of the Filoviridae family. Our initial approach was to construct hybrid VLPs containing heterologous viral proteins, of EBOV and MARV, and test the efficacy of the hybrid VLPs in a guinea pig model. Our data indicate that vaccination with GP was required and sufficient to protect against a homologous filovirus challenge, as heterologous wild-type VLPs or hybrid VLPs that did not contain the homologous GP failed to protect. Alternately, we vaccinated guinea pigs with a mixture of wild-type Ebola and Marburg VLPs. Vaccination with a single dose of the multivalent VLP vaccine elicited strong immune responses to both viruses and protected animals against EBOV and MARV challenge. This work provides a critical foundation towards the development of a pan-filovirus vaccine that is safe and effective for use in primates and humans.     

De novo syntheses of Marburg virus antigens from adenovirus vectors induce potent humoral and cellular immune responses

Marburg virus (MARV) is an African filovirus that causes a deadly hemorrhagic fever in humans, with up to 90% mortality. Currently, there are no MARV vaccines or therapies approved for human use. We hypothesized that developing a vaccine that induces a de novo synthesis of MARV antigens in vivo will lead to strong induction of both a humoral and cell-mediated immune response against MARV. Here, we develop and characterize three novel gene-based vaccine candidates which express the viral glycoprotein (GP) from either the Ci67, Ravn or Musoke strain of MARV. Immunization of mice with complex adenovirus (Ad)-based vaccine candidates (cAdVax vaccines), led to efficient production of both antibodies and cytotoxic T lymphocytes (CTL) specific to Musoke strain GP and Ci67 strain GP, respectively. Antibody responses were also shown to be cross-reactive across the MARV strains, but not cross-reactive to Ebola virus, a related filovirus. Additionally, three 1 x 10(8)pfu doses of vaccine vector were demonstrated to be safe in mice, as this did not lead to any detectable toxicity in liver or spleen. These promising results indicate that a cAdVax-based vaccine could be effective for induction of both humoral and cell-mediated immune responses to multiple strains of the Marburg virus.     

Long term immunity in African cattle vaccinated with a recombinant capripox-rinderpest virus vaccine

Cattle were vaccinated with a recombinant capripox-rinderpest vaccine designed to protect cattle from infection with either rinderpest virus (RPV) or lumpy skin disease virus (LSDV). Vaccination did not induce any adverse clinical responses or show evidence of transmission of the vaccine virus to in-contact control animals. Approximately 50% of the cattle were solidly protected from challenge with a lethal dose of virulent RPV 2 years after vaccination while at 3 years approx. 30% were fully protected. In the case of LSDV, all of 4 vaccinated cattle challenged with virulent LSDV at 2 years were completely protected from clinical disease while 2 of 5 vaccinated cattle were completely protected at 3 years. The recombinant vaccine showed no loss of potency when stored lyophylized at 4 degrees C for up to 1 year. These results indicate that capripoxvirus is a suitable vector for the development of safe, effective and stable recombinant vaccines for cattle.     

A recombinant vesicular stomatitis virus replicon vaccine protects chickens from highly pathogenic avian influenza virus (H7N1)

Highly pathogenic avian influenza viruses (HPAIV) of subtypes H5 and H7 cause fatal disease in poultry (fowl plague) but also have zoonotic potential. Currently commercially available vaccines often do not provide sufficient protection and do not allow easy discrimination between vaccinated and infected birds. Therefore, vaccination of domestic poultry against H5 and H7 HPAIV is not allowed in many countries, or is only possible after special permission has been provided. We generated a recombinant marker vaccine based on non-transmissible vesicular stomatitis virus (VSV) expressing the HA antigen of HPAIV A/FPV/Rostock/34 (H7N1) in place of the VSV G gene. This virus, VSV*ΔG(HA), was propagated on a helper cell line providing VSV G in trans. Since no progeny virus was produced after infection of non-complementing cells, the vector was classified as biosafety level 1 organism (“safe”). Chickens were immunized via the intramuscular route. Following booster vaccination with the same replicons high titers of serum antibodies were induced, which neutralized avian influenza viruses of subtypes H7N1 and H7N7 but not H5N2. Vaccinated chickens were protected against a lethal dose of heterologous HPAIV A/chicken/Italy/445/99 (H7N1). Secretion of challenge virus was short-term and significantly reduced. Finally, it was possible to discriminate vaccinated chickens from infected ones by a simple ELISA assay. We propose that VSV replicons have the potential to be developed to high-quality vaccines for protection of poultry against different subtypes of avian influenza viruses.     

Prospects,including time-frames,for improved foot and mouth disease vaccines

Inactivated foot and mouth disease (FMD) vaccines have been used successfully as part of eradication programmes. However, there are a number of concerns with the use of such vaccines and the recent outbreaks of FMD in disease-free countries have increased the need for improved FMD control strategies. To address this requirement, new generation FMD vaccines are being developed. Currently, one of the most promising of these vaccine candidates utilises an empty viral capsid subunit delivered to animals by a live virus vector. This candidate, a replication-defective recombinant human adenovirus containing the capsid and 3C proteinase coding regions of FMD virus (FMDV), induces an FMDV-specific neutralising antibody response in inoculated animals. Upon challenge with a virulent animal-passaged homologous virus, swine and cattle vaccinated with this recombinant adenovirus are protected from clinical signs of FMD as well as from FMDV replication. One inoculation of a high dose of this vaccine candidate protected swine from challenge as early as seven days after vaccination.     

Impact of prior flavivirus vaccination on immunogenicity and efficacy of an inactivated Zika vaccine in Indian rhesus macaques

Flaviviruses are antigenically related. We evaluated the immunogenicity and efficacy of Takeda’s purified inactivated Zika vaccine (PIZV) candidate in macaques previously vaccinated with several commercially available heterologous flavivirus vaccines. Heterologous flavivirus vaccination did not elicit Zika virus (ZIKV) neutralizing antibodies and did not impact neutralizing antibody titers after one dose of PIZV. After a second PIZV dose previous vaccination with flavivirus vaccines had variable impact on ZIKV neutralizing antibody titers. However, all macaques were protected against viremia after Zika virus challenge 8–12 months post-PIZV vaccination. Therefore, vaccine-induced immunity against heterologous flavivirus vaccines does not impact PIZV efficacy in macaques.     

Genetically engineered, live, attenuated vaccines protect nonhuman primates against aerosol challenge with a virulent IE strain of Venezuelan equine encephalitis virus

Two live, attenuated strains of Venezuelan equine encephalitis virus (VEE), IE1150K and V3526, were administered to macaques to determine if they could elicit protection against an aerosol challenge with virulent VEE virus of the IE variety (VEEV-IE). These viruses were rescued from full-length cDNA clones of 68U201 (VEEV-IE variety) and Trinidad donkey (VEEV-IA/B variety), respectively, and both have a furin cleavage site deletion mutation and a second-site resuscitating mutation. Both vaccines elicited neutralizing antibodies to viruses of the homologous variety but not to viruses of the heterologous variety. Eight weeks after vaccination, the macaques were challenged by aerosol exposure to virulent 68U201. Macaques vaccinated with V3526 were protected as well as macaques inoculated with IE1009, the wild-type infectious clone of 68U201. However, IE1150K failed to significantly protect macaques relative to controls. V3526 has now been shown to protect macaques against both IA/B [Pratt WD, Davis NL, Johnston RE, Smith JF. Genetically engineered, live attenuated vaccines for Venezuelan equine encephalitis: testing in animal models. Vaccine 2003;21(25-26):3854-62] and IE strains of VEE viruses.     

Protection in mice passively immunized with serum from cynomolgus macaques and humans vaccinated with recombinant plague vaccine (rF1V)

Passive transfer models were developed to evaluate the ability of antibodies generated in cynomolgus macaques and humans vaccinated with a recombinant plague vaccine (rF1V) to protect naïve Swiss Webster mice against pneumonic plague. Development of the passive transfer model is intended to support clinical and nonclinical development of the rF1V vaccine. To evaluate protection, unfractionated serum collected from rF1V vaccinated cynomolgus macaques and human volunteers with known antibody titers to rF1, rV and rF1V was transferred into naïve Swiss Webster mice via the intraperitoneal route. Results of these studies demonstrated that passive immunization protected mice from challenge or extended mean survival time and that the passive transfer assay can be used to evaluate the functional role of antibodies induced by rF1V vaccination in protection against aerosol exposure.     

Systematic review of Marburg virus vaccine nonhuman primate studies and human clinical trials

BackgroundRecent deadly outbreaks of Marburg virus underscore the need for an effective vaccine. A summary of the latest research is needed for this WHO priority pathogen. This systematic review aimed to determine progress towards a vaccine for Marburg virus.MethodsArticle search criteria were developed to query PubMed for peer-reviewed articles from 1990 through 2019 on Marburg virus vaccine clinical trials in humans and pre-clinical studies in non-human primates (NHP). Abstracts were reviewed by two authors. Relevant articles were reviewed in full. Discrepancies were resolved by a third author. Data abstracted included year, author, title, vaccine construct, number of subjects, efficacy, and demographics. Assessment for risk of bias was performed using the Syrcle tool for animal studies, and the Cochrane Collaboration risk of bias tool for human studies.Results101 articles were identified; 27 were related to Marburg vaccines. After full text review, 21 articles were selected. 215 human subjects were in three phase 1 clinical trials, and 203 NHP in 18 studies. Vaccine constructs were DNA plasmids, recombinant vesicular stomatitis virus (VSV) vectors, adenovirus vectors, virus-like particles (VLP), among others. Two human phase 1 studies of DNA vaccines had 4 adverse effects requiring vaccine discontinuation among 128 participants and 31–80% immunogenicity. In NHP challenge studies, 100% survival was seen in 6 VSV vectored vaccines, 2 DNA vaccines, 2 VLP vaccines, and in 1 adenoviral vectored vaccine.ConclusionIn human trials, two Marburg DNA vaccines provided either low immunogenicity or a failure to elicit durable immunity. A variety of NHP candidate Marburg vaccines demonstrated favorable survival and immunogenicity parameters, to include VSV, VLP, and adenoviral vectored vaccines. Elevated binding antibodies appeared to be consistently associated with protection across the NHP challenge studies. Further human trials are needed to advance vaccines to limit the spread of this highly lethal virus.     

Trial of a capripoxvirus-rinderpest recombinant vaccine in African cattle.

Cattle were vaccinated with differing doses of an equal mixture of capripox-rinderpest recombinant viruses expressing either the fusion protein (F) or the haemagglutinin protein (H) of rinderpest virus. Animals vaccinated with 2 x 10(4) p.f.u. or greater of the combined viruses were completely protected against challenge, 1 month later, with both virulent rinderpest and lumpy skin disease viruses. Vaccination with any of the doses did not induce any adverse clinical response in the animals or transmission of the vaccine virus between animals. All cattle challenged 6 or 12 months after vaccination with 2 x 10(5) p.f.u. of the mixture of recombinant viruses were protected from severe rinderpest disease. Ten out of 18 were completely protected while the remaining 8 developed mild clinical signs of rinderpest. Cattle vaccinated with the recombinant vaccines after prior infection with the parental capripox virus showed more marked clinical signs of rinderpest after challenge with virulent rinderpest, but 9 out of 10 recovered, compared with 80% mortality in the unvaccinated controls.     

Characterization of rVSVΔG-ZEBOV-GP glycoproteins using automated capillary western blotting

Ebolavirus (EBOV) entry to host cells requires membrane-associated glycoprotein (GP). A recombinant vesicular stomatitis virus vector carrying Zaire Ebola virus glycoprotein (rVSV-ZEBOV) was developed as a vaccine against ebolaviruses. The VSV glycoprotein gene was deleted (rVSVΔG) and ZEBOV glycoprotein (GP) was inserted into the deleted VSV glycoprotein open reading frame (ORF) resulting in a live, replication-competent vector (rVSVΔG-ZEBOV-GP). Automated capillary westerns were used to characterize the rVSVΔG-ZEBOV-GP vaccine (ERVEBO®) manufacturing process with regards to glycoprotein (GP) structure and variants. The method shows a unique electropherogram profile for each process step which could be used to monitor process robustness. rVSVΔG-ZEBOV-GP encodes GP (GP1-GP2), secreted GP (sGP), and small secreted GP (ssGP) variants. Furthermore, a TACE-like activity was observed indirectly by detecting soluble GP2Δ after virus precipitation by ultracentrifugation. Capillary western blotting techniques can guide process development by evaluating process steps such as enzyme treatment. In addition, the technique can assess GP stability and process lot-to-lot consistency. Finally, capillary western-based technology was used to identify a unique biochemical profile of the rVSVΔG-ZEBOV-GP vaccine strain in final product. Virion membrane-bound GP1-GP2 is critical to vaccine-elicited protection by providing both neutralizing antibodies and T-cell response.     

Yeast-expressed Puumala hantavirus nucleocapsid protein induces protection in a bank vole model

Hantaviruses are rodent-borne agents that cause severe human diseases. The coding sequences for the authentic and a His-tagged Puumala hantavirus (PUUV) nucleocapsid (N) protein were expressed in yeast (Saccharomyces cerevisiae). N-specific monoclonal antibodies demonstrated native antigenicity of the two proteins. All bank voles vaccinated with the His-tagged N protein in Freund's adjuvant (n=12) were defined as completely protected against subsequent virus challenge, based on the absence of viral N protein, RNA and G2-specific antibodies. In the group vaccinated with the yeast-expressed authentic N protein in Freund's adjuvant, 2/6 animals were defined as completely protected and 4/6 as partially protected. Moreover, when animals were vaccinated with the His-tagged N protein in an adjuvant certified for human use (alum), all (n=8) were at least partially protected (six completely, two partially). The general advantages of the yeast expression system make the described recombinant proteins promising candidate vaccines against hantavirus infection.     

Efficacy of a human anthrax vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin

The efficacy of a licensed human anthrax vaccine (Anthrax Vaccine Adsorbed (AVA)) was tested in guinea pigs, rabbits, and rhesus macaques against spore challenge by Bacillus anthracis isolates of diverse geographical origin. Initially, groups of Hartley guinea pigs were vaccinated at 0 and 4 weeks with AVA, then challenged intramuscularly at 10 weeks with spores from 33 isolates of B. anthracis. Survival among the vaccinated groups varied from 6 to 100%, although there were no differences in mean time to death among the groups. There was no correlation between isolate virulence and variable number tandem repeat category or protective antigen genotype identified. New Zealand white rabbits were then vaccinated with AVA at 0 and 4 weeks, and challenged at 10 weeks by aerosol with spores from six of the isolates that were highly virulent in vaccinated guinea pigs. AVA completely protected the rabbits from four of the isolates, and protected 90% of the animals from the other two isolates. Subsequently, two of these six isolates were then used to challenge rhesus macaques, previously vaccinated with AVA at 0 and 4 weeks, and challenged at 10 weeks by aerosol. AVA protected 80 and 100% of the animals from these two isolates. These studies demonstrated that, although AVA confers variable protection against different B. anthracis isolates in guinea pigs, it is highly protective against these same isolates in both rabbits and rhesus macaques.     

Efficacy of a B virus gD DNA vaccine for induction of humoral and cellular immune responses in Japanese macaques

It is desirable to prevent dissemination of B virus (BV) in macaque colonies because transmission of BV to humans causes deadly encephalomyelitis. Vaccination of monkeys is one method that could confine spread of BV within macaque colonies. Availability of a BV DNA vaccine for use in macaques would eliminate the risk of working with infectious BV. Toward this end, we constructed a plasmid expressing the BV glycoprotein D (gD). Immunogenicity of this construct as a DNA vaccine was assessed in adult Japanese macaques by four intracutaneous injections at a dose of 500 microg per head. Results of enzyme-linked immunosorbent assay (ELISA) using a recombinant herpes simplex virus type 1 (HSV1) gD, a homologue of BV gD, showed that significant levels of antibody was induced in all vaccinated animals following each booster injection. Western blot of sera from vaccinated macaques confirmed the specific recognition of authentic BV gD. Immune sera were also demonstrated to contain neutralizing activity against infectious BV. Weak lymphoproliferative responses were also observed in vaccinated macaques using recombinant HSV1 gD as a stimulating antigen and flow cytometry analysis of one individual revealed the presence of HSV1 gD-responsive effector T cells. Thus, the BV gD DNA vaccine was demonstrated to induce both humoral and cellular immune responses in macaques which recognized BV gD.     

Mucosal administration of three recombinant Mycobacterium bovis BCG-SIVmac251 strains to cynomolgus macaques induces rectal IgAs and boosts systemic cellular immune responses that are primed by intradermal vaccination

The widely administered Mycobacterium bovis BCG is an attractive live vector for the development of AIDS vaccines. We explored immune responses induced in cynomolgus macaques to rBCG-SIV(3), a mixture of three recombinant BCG strains expressing the SIVmac251 nef, gag and env genes. After a single intradermal (ID) inoculation, circulating blood cells from rBCG-SIV(3)-vaccinated monkeys exhibited CTL responses targeted against the three antigens and interferon-gamma (IFNgamma) secretion was observed. A rectal or oral boosting dose of rBCG-SIV(3) elicited anti-SIV IgAs in the rectum of vaccinated monkeys and increased IFNgamma secretion by circulating blood cells. Despite a good response against the vector, rBCG-SIV(3) administration did not induce IgG antibody responses or lymphoproliferation against the SIV antigens in blood. This could be due to the lack of in vivo persistence of the recombinant BCG strains that were used. Rectal challenge with fully pathogenic SIVmac251-infected all animals. However, after viral challenge, anti-SIV cellular and antibody responses were higher in rBCG-SIV(3) monkeys than in controls indicating that the vaccine induced anti-SIV CD4(+) T-cell memory.     

Efficacy of a live attenuated tetravalent candidate dengue vaccine in naïve and previously infected cynomolgus macaques

The development of a safe and effective vaccine against dengue is a public health priority. Attempts to evaluate candidate vaccine formulations in human volunteers were largely unsuccessful, at least in part due to too high reactogenicity of some of the candidate vaccines tested. We evaluated a live attenuated tetravalent dengue vaccine candidate in flavivirus na?ve and dengue virus type 3 immune non-human primates. Immune responses were measured both at the humoral and the cellular level and the efficacy of this vaccine candidate was evaluated by challenging the vaccinated animals with dengue virus type 4. Humoral and cellular immune responses upon vaccination were similar to those described after natural infection in humans. All animals were protected from developing viremia upon challenge infection. In addition, primary dengue virus type 3 infection of macaques neither influenced the immune response upon vaccination, nor interfered with vaccine-induced protection from dengue virus type 4 challenge infection. The data suggest that the live attenuated tetravalent vaccine candidate used is promising and warrant further safety and efficacy testing in clinical trials.     

Postexposure Treatment of Marburg Virus Infection

Rhesus monkeys are protected from disease when a recombinant vesicular stomatitis virus–based vaccine is administered 20–30 min after infection with Marburg virus. We protected 5/6 monkeys when this vaccine was given 24 h after challenge; 2/6 animals were protected when the vaccine was administered 48 h postinfection.