A cytomegalovirus-based vaccine provides long-lasting protection against lethal Ebola virus challenge after a single dose

Ebola virus (Zaire ebolavirus; EBOV) is a highly lethal hemorrhagic disease virus that most recently was responsible for two independent 2014 outbreaks in multiple countries in Western Africa, and the Democratic Republic of the Congo, respectively. Herein, we show that a cytomegalovirus (CMV)-based vaccine provides durable protective immunity from Ebola virus following a single vaccine dose. This study has implications for human vaccination against ebolaviruses, as well as for development of a ‘disseminating’ vaccine to target these viruses in wild African great apes.     

Chimeric alphavirus vaccine candidates protect mice from intranasal challenge with western equine encephalitis virus

We developed two types of chimeric Sindbis virus (SINV)/western equine encephalitis virus (WEEV) alphaviruses to investigate their potential use as live virus vaccines against WEE. The first-generation vaccine candidate, SIN/CO92, was derived from structural protein genes of WEEV strain CO92-1356, and two second-generation candidates were derived from WEEV strain McMillan. For both first- and second-generation vaccine candidates, the nonstructural protein genes were derived from SINV strain AR339. Second-generation vaccine candidates SIN/SIN/McM and SIN/EEE/McM included the envelope glycoprotein genes from WEEV strain McMillan; however, the amino-terminal half of the capsid, which encodes the RNA-binding domain, was derived from either SINV or eastern equine encephalitis virus (EEEV) strain FL93-939. All chimeric viruses replicated efficiently in mammalian and mosquito cell cultures and were highly attenuated in 6-week-old mice. Vaccinated mice developed little or no detectable disease and showed little or no evidence of challenge virus replication; however, all developed high titers of neutralizing antibodies. Upon intranasal challenge with high doses of virulent WEEV strains, mice vaccinated with ≥10⁵ PFU of SIN/CO92 or ≥10⁴ PFU of SIN/SIN/McM or SIN/EEE/McM were completely protected from disease. These findings support the potential use of these live-attenuated vaccine candidates as safe and effective vaccines against WEE.     

A chimeric Sindbis-based vaccine protects cynomolgus macaques against a lethal aerosol challenge of eastern equine encephalitis virus

Eastern equine encephalitis virus (EEEV) is a mosquito-borne alphavirus that causes sporadic, often fatal disease outbreaks in humans and equids, and is also a biological threat agent. Two chimeric vaccine candidates were constructed using a cDNA clone with a Sindbis virus (SINV) backbone and structural protein genes from either a North (SIN/NAEEEV) or South American (SIN/SAEEEV) strain of EEEV. The vaccine candidates were tested in a nonhuman primate (NHP) model of eastern equine encephalitis (EEE). Cynomolgus macaques were either sham-vaccinated, or vaccinated with a single dose of either SIN/NAEEEV or SIN/SAEEEV. After vaccination, animals were challenged by aerosol with a virulent North American strain of EEEV (NA EEEV). The SIN/NAEEEV vaccine provided significant protection, and most vaccinated animals survived EEEV challenge (82%) with little evidence of disease, whereas most SIN/SAEEEV-vaccinated (83%) and control (100%) animals died. Protected animals exhibited minimal changes in temperature and cardiovascular rhythm, whereas unprotected animals showed profound hyperthermia and changes in heart rate postexposure. Acute inflammation and neuronal necrosis were consistent with EEEV-induced encephalitis in unprotected animals, whereas no encephalitis-related histopathologic changes were observed in the SIN/NAEEEV-vaccinated animals. These results demonstrate that the chimeric SIN/NAEEEV vaccine candidate protects against an aerosol EEEV exposure.     

A recombinant humanized monoclonal antibody completely protects mice against lethal challenge with Venezuelan equine encephalitis virus

A recombinant humanized antibody to Venezuelan equine encephalitis virus (VEEV) was constructed in a monocistronic adenoviral expression vector with a foot-and-mouth-disease virus-derived 2A self-cleavage oligopeptide inserted between the antibody heavy and light chains. After expression in mammalian cells, the heavy and light chains of the humanized antibody (hu1A4A1IgG1-2A) were completely cleaved and properly dimerized. The purified hu1A4A1IgG1-2A retained VEEV binding affinity and neutralizing activity similar to its parental murine antibody. The half-life of hu1A4A1IgG1-2A in mice was approximately 2 days. Passive immunization of hu1A4A1IgG1-2A in mice (50 μg/mouse) 24 h before or after virulent VEEV challenge provided complete protection, indicating that hu1A4A1IgG1-2A has potent prophylactic and therapeutic effects against VEEV infection.     

Single-dose, fast-acting vaccine candidate against western equine encephalitis virus completely protects mice from intranasal challenge with different strains of the virus

Western equine encephalitis virus (WEEV) causes a fatal infection of the central nervous system in humans and horses. However, neither human vaccine nor antiviral drug is available. We found previously that immunization of mice with two doses of an adenovirus-vectored WEEV vaccine, Ad5-WEEV, confers complete protection against homologous WEEV challenge. In this paper, we report that a single-dose injection of Ad5-WEEV completely protected mice against both homologous and heterologous strains of WEEV at 1 week after immunization. In addition, mice immunized with Ad5-WEEV were protected when challenged at 13 weeks after a single-dose immunization. Therefore, the protection conferred by Ad5-WEEV is rapid, cross-protective, and long-lasting. These results warrant further development of Ad5-WEEV into an emergency vaccine that can be used during a natural outbreak or a bioterrorism attack.     

Complete protection against lethal challenge of novel H7N9 virus with heterologous inactivated H7 vaccine in mice

A prototype H7 influenza vaccine constructed based on the H7N7 outbreak in 2003 was tested for the protective efficacy against the novel H7N9 virus in a lethal murine challenge model. Serum samples from vaccinated mice showed significant neutralizing activity against the H7N9 virus and the mice were completely protected with no significant weight loss. The results have direct implications on how to overcome potential vaccine shortage and identify donors for immune sera for passive immunization.     

Enhanced protection against a lethal influenza virus challenge by immunization with both hemagglutinin- and neuraminidase-expressing DNAs

The ability of plasmid DNA encoding hemagglutinin (HA), neuraminidase (NA) or matrix protein (M1) from influenza virus A/PR/8/34 (PR8) (H1N1), and mixtures of these plasmid DNAs (HA + NA and HA + NA + M1) to protect against homologous or heterologous virus infection was examined in BALB/c mice. Each DNA was inoculated twice, 3 weeks apart, or four times, 2 weeks apart, at a dose of 1 microg of each component per mouse by particle-mediated DNA transfer to the epidermis (gene gun). Seven days after the last immunization, mice were challenged with a lethal homologous or heterologous virus and the ability of each DNA to protect the mice from influenza was evaluated by observing lung virus titers and survival rates. The administration of a plasmid DNA mixture of either (HA + NA) or (HA + NA + M1) provided almost complete protection against the PR8 virus challenge, and this protection was accompanied by high levels of specific antibody responses to the respective components. The degree of protection afforded in these groups is significantly higher than that in mice given either HA- or NA-expressing DNA alone, which provided only a partial protection against PR8 challenge or that in mice given M1-expressing DNA, which failed to provide any protection. In addition, both of the plasmid DNA mixtures (HA + NA) and (HA + NA + M1) showed a slight tendency to provide cross-protection against an A/Yamagata/120/86 (H1N1) virus challenge, and this was accompanied by a relatively high level of cross-reacting antibodies. Thus, there was no clear difference between the ability of the HA + NA and HA + NA + M1 plasmid DNA mixtures in providing protection against either a PR8 or heterologous virus challenge. These results suggest that in mice immunized by gene gun, a mixture of plasmid DNAs encoding HA and NA can provide the most effective protection against the virus challenge. The addition of the M -expressing plasmid DNA to this mixture does not enhance the degree of protection afforded.     

CD4 T cells provide protection against acute lethal encephalitis caused by Venezuelan equine encephalitis virus

Studying the mechanisms of host survival resulting from viral encephalitis is critical to the development of vaccines. Here we have shown in several independent studies that high dose treatment with neutralizing antibody prior to intranasal infection with Venezuelan equine encephalitis virus had an antiviral effect in the visceral organs and prolonged survival time of infected mice, even in the absence of αβ T cells. Nevertheless, antibody treatment did not prevent the development of lethal encephalitis. On the contrary, the adoptive transfer of primed CD4⁺ T cells was necessary to prevent lethal encephalitis in mice lacking αβ T cell receptor.     

Immunity to airborne challenge with Venezuelan equine encephalitis virus develops rapidly after immunization with the attenuated vaccine strain TC-83.

Mice vaccinated subcutaneously with the attenuated vaccine strain of Venezuelan equine encephalitis virus (VEEV) rapidly develop immunity to subcutaneous or airborne challenge with virulent VEEV. The specificity of this immune response was demonstrated by challenge with a heterologous virus (St. Louis encephalitis virus). Examination of the levels of VEEV-specific antibody classes in serum and respiratory secretions suggested that the rapid development of immunity was coincident with the appearance of specific IgM and IgG (but not IgA) in the respiratory tract. In order to confirm the role of respiratory tract antibody, mice were passively immunised either intraperitoneally or intranasally with polyclonal VEEV-specific IgG. Intranasal administration of specific IgG significantly enhanced protection against airborne challenge. These results confirm the need to emphasise local antibody production in the development of improved VEEV vaccines.     

ISCOM vaccine induced protection against a lethal challenge with a human H5N1 influenza virus

Recently avian influenza A viruses of the H5N1 subtype were shown to infect humans in the Hong Kong area, resulting in the death of six people. Although these viruses did not efficiently spread amongst humans, these events illustrated that influenza viruses of subtypes not previously detected in humans could be at the basis of a new pandemic. In the light of this pandemic threat we evaluated and compared the efficacy of a classical non-adjuvanted subunit vaccine and a vaccine based on immune stimulating complexes (ISCOM) prepared with the membrane glycoproteins of the human influenza virus A/Hong Kong 156/97 (H5N1) to protect roosters against a lethal challenge with this virus. The ISCOM vaccine induced protective immunity against the challenge infection whereas the non-adjuvanted subunit vaccine proved to be poorly immunogenic and failed to induce protection in this model.     

Intranasal inoculate of influenza virus vaccine against lethal virus challenge

Vaccine adjuvants are essential for enhancing immune responses during vaccination. However, only a limited number of safe and effective adjuvants, especially mucosal adjuvants, are available for use in vaccines. The development of a practically applicable mucosal adjuvant is therefore urgently needed. Here, we showed that the non-toxic CTA1-DD adjuvant, which combined the full enzymatic activity of the A1 subunit of cholera toxin (CT) with two immunoglobulin-binding domains of Staphylococcus aureus protein A (SpA), promoted mucosal and systemic humoral and cell-mediated immune responses following intranasal administration with H1N1 split vaccine in mice. We demonstrated that CTA1-DD-adjuvant vaccine provided 100% protection against mortality and greatly reduced morbidity in a mouse model. We also showed that addition of CTA1-DD to the vaccine elicited significantly higher hemagglutination inhibition titers and IgG antibodies in sera than alum adjuvant. Furthermore, CTA1-DD significantly promoted the production of mucosal secretory IgA in lung lavages and vaginal lavages. We also showed that CTA1-DD could be used as a mucosal adjuvant to enhance T cell responses. Our results clearly indicated that CTA1-DD contributed to the elicitation of a protective cell-mediated immune response required for efficacious vaccination against influenza virus, which suggested that this adjuvant could be explored further as a clinically safe mucosal vaccine adjuvant for respiratory diseases and other mucosal diseases.     

Protective immunity against botulism provided by a single dose vaccination with an adenovirus-vectored vaccine

Botulinum neurotoxins cause botulism, a neuroparalytic disease in humans and animals. We constructed a replication-incompetent adenovirus encoding a synthesized codon-optimized gene for expression of the heavy chain C-fragment (H(C)50) of botulinum neurotoxin type C (BoNT/C). This recombinant human serotype 5 adenoviral vector (Ad5) was evaluated as a genetic vaccine candidate against botulism caused by BoNT/C in a mouse model. A one-time intramuscular injection with 10(5) to 2 x 10(7)pfu of adenoviral vectors elicited robust serum antibody responses against H(C)50 of BoNT/C as assessed by ELISA. Immune sera showed high potency in neutralizing the active BoNT/C in vitro. After a single dose of 2 x 10(7)pfu adenoviral vectors, the animals were completely protected against intraperitoneal challenge with 100 x MLD(50) of active BoNT/C. The protective immunity appeared to be vaccine dose-dependent. The anti-toxin protective immunity could last for at least 7 months without a booster injection. In addition, we observed that pre-existing immunity to the wild-type Ad5 in the host had no significant influence on the protective efficacy of vaccination. The data suggest that an adenovirus-vectored genetic vaccine is a highly efficient prophylaxis candidate against botulism.     

A virus-like particle vaccine confers protection against enterovirus D68 lethal challenge in mice

Enterovirus D68 (EV-D68) is increasingly associated with severe acute respiratory infection and acute flaccid myelitis (AFM) in children around the world. However, neither vaccines nor therapeutic drugs are available for EV-D68. Here we report the development of a virus-like particle (VLP) based experimental EV-D68 vaccine. We found that EV-D68 VLPs could be successfully generated in insect cells infected with a recombinant baculovirus co-expressing the P1 precursor and 3CD protease of EV-D68. Biochemical and electron microscopic analyses revealed that EV-D68 VLPs were composed of VP0, VP1, and VP3 capsid proteins derived from precursor P1 and were visualized as spherical particles of ∼30 nm in diameter. Immunization of mice with EV-D68 VLPs resulted in the production of serum antibodies that displayed potent serotype-specific neutralizing activities against EV-D68 virus in vitro. Passive transfer of anti-VLP sera completely protected neonatal recipient mice from lethal EV-D68 infection. Moreover, maternal immunization with these VLPs provided full protection against lethal EV-D68 challenge in suckling mice. Together, these results demonstrate that the recombinant EV-D68 VLP is a promising vaccine candidate against EV-D68 infection.     

Serum albumin nanoparticles vaccine provides protection against a lethal Pseudomonas aeruginosa challenge

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections in immunocompromised individuals and in patients with cystic fibrosis. A range of vaccines to prevent infections caused by P. aeruginosa has already been tested, yet no vaccine against this pathogen is currently available. The goal of this study was to evaluate the potential of bovine serum albumin nanoparticles (BSA-NPs) associated with total P. aeruginosa ATCC 27853 antigens in inducing protection against the infection with virulent P. aeruginosa PA14 strain in murine model of nasal infection. Swiss mice were immunized with BSA-NPs associated with total P. aeruginosa antigens (NPPa) or empty NPs (NPe). As positive and negative control, groups of animals were immunized with total antigens of P. aeruginosa ATCC 27853 and phosphate buffered saline, respectively. Immunized mice were infected via nasal route using P. aeruginosa PA14 strain. The survival after 48 h was evaluated and the lungs from animals were processed for quantification of bacterial load, cytokine expression and histopathological analysis. After infection with P. aeruginosa PA14, animals immunized with NPPa had the highest survival rate, the lowest bacterial lung load, a controlled production of cytokines and few histopathological changes. These results indicate that NPPa immunization protected mice from infection, contributing for the elimination of the bacteria from the lungs, which consequently reflected the survival of the animals. Therefore, this vaccine was able to induce a functional response in an animal model of lethal infection and thereby is a promising platform for P. aeruginosa vaccines.     

A single immunization with a dry powder anthrax vaccine protects rabbits against lethal aerosol challenge

Here we confirm that intranasal (IN) dry powder anthrax vaccine formulations are able to protect rabbits against aerosol challenge 9 weeks after a single immunization. The optimum dose of rPA in our dry powder anthrax vaccine formulation in rabbits was experimentally determined to be 150microg and therefore was chosen as the target dose for all subsequent experiments. Rabbits received a single dose of either 150microg rPA, 150microg rPA+150microg of a conjugated 10-mer peptide representing the Bacillus anthracis capsule (conj), or 150microg of conj alone. All dry powder formulations contained MPL and chitosan (ChiSys). Significant anti-rPA titers and anthrax lethal toxin neutralizing antibody (TNA) levels were seen with both rPA containing vaccines, although rPA-specific IgG and TNA levels were reduced in rabbits immunized with rPA plus conj. Nine weeks after immunization, rabbits were exposed to a mean aerosol challenge dose of 278 LD50 of Ames spores. Groups immunized with rPA or with rPA+conj had significant increases in survivor proportions compared to the negative control group by Logrank test (p=0.0001 and 0.003, respectively), and survival was not statistically different for the rPA and rPA+conj immunized groups (p=0.63). These data demonstrate that a single immunization with our dry powder anthrax vaccine can protect against a lethal aerosol spore challenge 9 weeks later.     

Intramuscular immunization with a vesicular stomatitis virus recombinant expressing the influenza hemagglutinin provides post-exposure protection against lethal influenza challenge

Vaccines currently licensed for the prevention of seasonal influenza induce antibodies against the influenza hemagglutinin (HA) and neuraminidase (NA) contained in the vaccine preparation but require at least 2 weeks after immunization for the development of protective immunity. These vaccines do not induce protective responses quickly enough to blunt the effects of infection when administered after exposure. We have developed a novel vaccine based on recombinant vesicular stomatitis virus which expresses the influenza hemagglutinin (rVSV HA) and protects mice from lethal influenza challenge when the vaccine is administered intramuscularly at least 24 h after delivery of the influenza challenge virus. To our knowledge this is the first vaccine that effectively protects animals from lethal influenza challenge when delivered by a systemic route after influenza exposure has occurred. The induction of HA-specific immune responses by the vaccine is necessary for full protection from challenge, because animals immunized with an empty rVSV vector were not protected equally. Our results are consistent with a model in which vaccination induces an immediate antiviral cytokine response, followed by development of humoral and cellular immune responses which act to reduce pulmonary viral loads and accelerate recovery. Consistent with this model, mice vaccinated with the specific vaccine rVSV HA had high levels of IFN-α in the serum by 24 h after challenge/vaccination, developed serum neutralizing Ab to influenza 2 days prior to control animals, and had detectable anti-HA CD8 T cells present in the peripheral blood 3 days prior to control mice.     

Immunogenicity and protective efficacy of a DNA vaccine against Venezuelan equine encephalitis virus aerosol challenge in nonhuman primates

A study to evaluate the immunogenicity and protective efficacy of a Venezuelan equine encephalitis virus (VEEV) DNA vaccine in an aerosol model of nonhuman primate infection was performed. Cynomolgus macaques vaccinated with a plasmid expressing the 26S structural genes of VEEV subtype IAB by particle-mediated epidermal delivery (PMED) developed virus-neutralizing antibodies. No serum viremia was detected in two out of three macaques vaccinated with the VEEV DNA after aerosol challenge with homologous virus, while one displayed a low viremia on a single day postchallenge. In contrast, all three macaques vaccinated with empty vector DNA developed a high viremia that persisted for at least 3 days after challenge. In addition, macaques vaccinated with the VEEV DNA had reduced febrile reactions, lymphopenia, and clinical signs of disease postchallenge as compared to negative control macaques. Therefore, although the sample size was small in this pilot study, these results indicate that a VEEV DNA vaccine administered by PMED can at least partially protect nonhuman primates against an aerosol VEEV challenge.     

TC-83 vaccine protects against airborne or subcutaneous challenge with heterologous mouse-virulent strains of Venezuelan equine encephalitis virus

Vaccination with TC-83 virus produced solid protection against subcutaneous challenge with Venezuelan equine encephalitis (VEEV) viruses from homologous and heterologous serogroups, but breakthrough infection and disease occurred after airborne challenge. Breakthrough occurred more often with time after vaccination, and was more frequent with epizootic, homologous serogroup 1A/B viruses than with enzootic, heterologous serogroup viruses. A decrease in VEEV-specific IgA levels in the respiratory tract of vaccinated mice may explain the increased frequency of breakthrough with time after vaccination. However increased breakthrough with the highly virulent homologous serogroup 1A/B viruses (compared to less virulent viruses from heterologous serogroups) may be a consequence of their greater ability to invade the brain via the olfactory neuroepithelium and olfactory nerve.