Novel DNA-launched Venezuelan equine encephalitis virus vaccine with rearranged genome

Novel live-attenuated V4020 vaccine was prepared for Venezuelan equine encephalitis virus (VEEV), an alphavirus from the Togaviridae family. The genome of V4020 virus was rearranged, with the capsid gene expressed using a duplicate subgenomic promoter downstream from the glycoprotein genes. V4020 also included both attenuating mutations from the TC83 VEEV vaccine secured by mutagenesis to prevent reversion mutations. The full-length infectious RNA of V4020 vaccine virus was expressed from pMG4020 plasmid downstream from the CMV promoter and launched replication of live-attenuated V4020 in vitro or in vivo. BALB/c mice vaccinated with a single dose of V4020 virus or with pMG4020 plasmid had no adverse reactions to vaccinations and developed high titers of neutralizing antibodies. After challenge with the wild type VEEV, vaccinated mice survived with no morbidity, while all unvaccinated controls succumbed to lethal infection. Intracranial injections in mice showed attenuated replication of V4020 vaccine virus as compared to the TC83. We conclude that V4020 vaccine has safety advantage over TC83, while provides equivalent protection in a mouse VEEV challenge model.     

A recombinant measles vaccine expressing chikungunya virus-like particles is strongly immunogenic and protects mice from lethal challenge with chikungunya virus

Chikungunya virus (CHIKV), a mosquito-transmitted alphavirus, recently reemerged in the Indian Ocean, India and Southeast Asia, causing millions of cases of severe polyarthralgia. No specific treatment to prevent disease or vaccine to limit epidemics is currently available. Here we describe a recombinant live-attenuated measles vaccine (MV) expressing CHIKV virus-like particles comprising capsid and envelope structural proteins from the recent CHIKV strain La Reunion. Immunization of mice susceptible to measles virus induced high titers of CHIKV antibodies that neutralized several primary isolates. Specific cellular immune responses were also elicited. A single immunization with this vaccine candidate protected all mice from a lethal CHIKV challenge, and passive transfer of immune sera conferred protection to naïve mice. Measles vaccine is one of the safest and most effective human vaccines. A recombinant MV-CHIKV virus could make a safe and effective vaccine against chikungunya that deserves to be further tested in human trials.     

Protection against avian coronavirus conferred by oral vaccination with live bacteria secreting LTB-fused viral proteins

The devastating impact of infectious bronchitis (IB) triggered by the IB virus (IBV), on poultry farms is generally curbed by livestock vaccination with live attenuated or inactivated vaccines. Yet, this approach is challenged by continuously emerging variants and by time limitations of vaccine preparation techniques. This work describes the design and evaluation of an anti-IBV vaccine comprised of E. coli expressing and secreting viral spike 1 subunit (S1) and nucleocapsid N-terminus and C-terminus polypeptides fused to heat-labile enterotoxin B (LTB) (LS1, LNN, LNC, respectively). Following chicken oral vaccination, anti-IBV IgY levels and cellular-mediated immunity as well as protection against virulent IBV challenge, were evaluated 14 days following the booster dose. Oral vaccination induced IgY levels that exceeded those measured following vaccination with each component separately. Following exposure to inactivated IBV, splenocytes isolated from chicks orally vaccinated with LNN or LNC -expressing bacteria, showed a higher percentage of CD8⁺ cells as compared to splenocytes isolated from chicks vaccinated with wild type or LTB-secreting E. coli and to chicks subcutaneously vaccinated. Significant reduction in viral load and percent of shedders in the vaccinated chicks was evident starting 3 days following challenge with 10^7.5 EID₅₀/ml virulent IBV. Taken together, orally delivered LTB-fused IBV polypeptide-expressing bacteria induced virus-specific IgY antibody production and was associated with significantly shorter viral shedding on challenge with a live IBV. The proposed vaccine design and delivery route promise an effective and rapidly adaptable means of protecting poultry farms from devastating IB outbreaks.     

Cross-protective immunity against o'nyong-nyong virus afforded by a novel recombinant chikungunya vaccine

Emerging mosquito-borne alphavirus infections caused by chikungunya virus (CHIKV) or o'nyong-nyong virus (ONNV) are responsible for sporadic and sometimes explosive urban outbreaks. Currently, there is no licensed vaccine against either virus. We have developed a highly attenuated recombinant CHIKV candidate vaccine (CHIKV/IRES) that in preclinical studies was demonstrated to be safe, immunogenic and efficacious. In this study we investigated the potential of this vaccine to induce cross-protective immunity against the antigenically related ONNV. Our studies demonstrated that a single dose of CHIKV/IRES elicited a strong cross-neutralizing antibody response and conferred protection against ONNV challenge in the A129 mouse model. Moreover, CHIKV/IRES immune A129 dams transferred antibodies to their offspring that were protective, and passively transferred anti-CHIKV/IRES immune serum protected AG129 mice, independently of a functional IFN response. These findings highlight the potential of the CHIKV/IRES vaccine to protect humans against not only CHIKV but also against ONNV-induced disease.     

Seroprevalence of Antibodies against Chikungunya,Dengue, and Rift Valley Fever Viruses after Febrile Illness Outbreak,Madagascar

In October 2009, two–3 months after an outbreak of a febrile disease with joint pain on the eastern coast of Madagascar, we assessed serologic markers for chikungunya virus (CHIKV), dengue virus (DENV), and Rift Valley fever virus (RVFV) in 1,244 pregnant women at 6 locations. In 2 eastern coast towns, IgG seroprevalence against CHIKV was 45% and 23%; IgM seroprevalence was 28% and 5%. IgG seroprevalence against DENV was 17% and 11%. No anti-DENV IgM was detected. At 4 locations, 450–1,300 m high, IgG seroprevalence against CHIKV was 0%–3%, suggesting CHIKV had not spread to higher inland-altitudes. Four women had IgG against RVFV, probably antibodies from a 2008 epidemic. Most (78%) women from coastal locations with CHIKV-specific IgG reported joint pain and stiffness; 21% reported no symptoms. CHIKV infection was significantly associated with high bodyweight. The outbreak was an isolated CHIKV epidemic without relevant DENV co-transmission.     

Rewiring of one carbon metabolism in Salmonella serves as an excellent live vaccine against systemic salmonellosis

Live attenuated vaccines are superior to the killed or subunit vaccines. We designed a Salmonella Typhimurium strain by deleting folD gene (encoding methylenetetrahydrofolate dehydrogenase-cyclohydrolase) in the presence of a heterologous fhs gene (encoding formyltetrahydrofolate synthetase) and tested its vaccine potential under stringent conditions of lethal and sub-lethal challenges with virulent Salmonella in the murine model. The efficacy of the vaccine in conferring protection against Salmonella infection was determined in a wide range of host conditions of systemic infection, corresponding to human young adults, neonates, geriatric age and, importantly, to the immune compromised state of pregnancy. The standardized vaccination regime comprised a primary dose of 10⁴ CFU/animal followed by a booster dose of 10² CFU/animal on day 7. Challenge with the virulent pathogen was done at day 7 post-administration of the booster. Subsequently, the mortality, morbidity, systemic colonization, antibody response and cytokine profiling were determined. The vaccinated cohort showed a strong protection against virulent pathogen in all models tested. The serum anti-Salmonella antibody titers and cytokine levels were significantly higher in the vaccinated cohort compared to the mock vaccinated cohort. Thus, we report the development and validation of a live attenuated vaccine candidate conferring excellent protection against Salmonellosis and typhoid fever.     

A novel liposomal adjuvant system,CAF01, promotes long-lived Mycobacterium tuberculosis-specific T-cell responses in human

Here, we report on a first-in-man trial where the tuberculosis (TB) vaccine Ag85B-ESAT-6 (H1) was adjuvanted with escalating doses of a novel liposome adjuvant CAF01. On their own, protein antigens cannot sufficiently induce immune responses in humans, and require the addition of an adjuvant system to ensure appropriate delivery and concomitant immune activation. To date no approved adjuvants are available for induction of cellular immunity, which seems essential for a number of vaccines, including vaccines against TB. We vaccinated four groups of human volunteers: a non-adjuvanted H1 group, followed by three groups with escalating doses of CAF01-adjuvanted H1 vaccine. All subjects were vaccinated at 0 and 8 weeks and followed up for 150 weeks. Vaccination did not cause local or systemic adverse effects besides transient soreness at the injection site. Two vaccinations elicited strong antigen-specific T-cell responses which persisted after 150 weeks follow-up, indicating the induction of a long-lasting memory response in the vaccine recipients. These results show that CAF01 is a safe and tolerable, Th1-inducing adjuvant for human TB vaccination trials and for vaccination studies in general where cellular immunity is required.     

Protective duration of immunity of an inactivated bluetongue (BTV) serotype 2 vaccine against a virulent BTV serotype 2 challenge in sheep

The protective properties of an inactivated bluetongue virus serotype 2 (BTV-2) vaccine were evaluated in sheep. Sheep (two groups of seven), vaccinated with either one or two doses of the vaccine, were monitored for antibody response over one year. All sheep developed high titres of neutralizing antibodies by 35 days after first vaccination and titres were maintained over one year. Control sheep (n = 7) remained seronegative until challenge. One year after vaccination, all sheep were inoculated with a virulent BTV-2. All controls developed pyrexia, clinical signs and viraemia. In contrast, the sheep vaccinated with one or two doses of inactivated BTV-2 vaccine were protected from clinical disease and viraemia was completely prevented. These data show that a single dose of the BTV-2 vaccine given to sheep induces a strong immunity which confers protection for at least one year.     

Evaluation of synthetic infection-enhancing lipopeptides as adjuvants for a live-attenuated canine distemper virus vaccine administered intra-nasally to ferrets

Background

Inactivated paramyxovirus vaccines have been associated with hypersensitivity responses upon challenge infection. For measles and canine distemper virus (CDV) safe and effective live-attenuated virus vaccines are available, but for human respiratory syncytial virus and human metapneumovirus development of such vaccines has proven difficult. We recently identified three synthetic bacterial lipopeptides that enhance paramyxovirus infections in vitro, and hypothesized these could be used as adjuvants to promote immune responses induced by live-attenuated paramyxovirus vaccines.

Methods

Here, we tested this hypothesis using a CDV vaccination and challenge model in ferrets. Three groups of six animals were intra-nasally vaccinated with recombinant (r) CDV^5804PL(CCEGFPC) in the presence or absence of the infection-enhancing lipopeptides Pam3CSK4 or PHCSK4. The recombinant CDV vaccine virus had previously been described to be over-attenuated in ferrets. A group of six animals was mock-vaccinated as control. Six weeks after vaccination all animals were challenged with a lethal dose of rCDV strain Snyder-Hill expressing the red fluorescent protein dTomato.

Results

Unexpectedly, intra-nasal vaccination of ferrets with rCDV^5804PL(CCEGFPC) in the absence of lipopeptides resulted in good immune responses and protection against lethal challenge infection. However, in animals vaccinated with lipopeptide-adjuvanted virus significantly higher vaccine virus loads were detected in nasopharyngeal lavages and peripheral blood mononuclear cells. In addition, these animals developed significantly higher CDV neutralizing antibody titers compared to animals vaccinated with non-adjuvanted vaccine.

Conclusions

This study demonstrates that the synthetic cationic lipopeptides Pam3CSK4 and PHCSK4 not only enhance paramyxovirus infection in vitro, but also in vivo. Given the observed enhancement of immunogenicity their potential as adjuvants for other live-attenuated paramyxovirus vaccines should be considered.     

Persistence of long-term immunity to hepatitis B among adolescents immunized at birth

The long-term duration of recombinant hepatitis B vaccine-induced immunity among persons vaccinated starting at birth is still not well understood. Waning of vaccine-induced immunity could leave young adults at risk of hepatitis B virus infection due to behavioral or occupational exposures. We followed a cohort of children immunized starting at birth with a 3-dose regimen of recombinant hepatitis B vaccine (5 mcg, 2.5 mcg, 2.5 mcg). They were challenged with a booster dose of the hepatitis B vaccine 10 and 15 years after vaccination to assess anamnestic response as a measure of persistence of protection. Among 108 participants who had lost protective antibody levels against hepatitis B, the majority (>70%) had an anamnestic response to the booster dose; response rates did not decline significantly between 10 and 15 years follow-up periods. A high antibody concentration following primary vaccination was independently associated with an anamnestic response later on in life. Nonetheless, ~20-30% of participants were unable to mount an immune response after boosting. Hepatitis B revaccination might be required for persons vaccinated starting at birth if opportunities for hepatitis B virus exposure exist. Future vaccine recommendations should be based on studies ascertaining protection against clinically significant disease.     

Evaluation of Japanese encephalitis virus E and NS1 proteins immunogenicity using a recombinant Newcastle disease virus in mice

Japanese encephalitis (JE) is the most important cause of acute encephalitis syndrome (AES). Japanese encephalitis virus (JEV), the prototype member of the JE serocomplex, belongs to the genus Flavivirus. The immunogenic proteins envelope (E) and non-structural protein 1 (NS1) of JEV are widely explored for the development of vaccines and diagnostics against JEV. However, there are underlying concerns such as the risk of reversion of live-attenuated vaccines to high virulence, the incomplete inactivation of pathogens in inactivated vaccines and partial vaccine coverage. Newcastle disease virus (NDV) is an efficient viral vaccine vector to express several human and animal immunogenic proteins. In the present study, we have developed a recombinant NDV (rNDV), individually expressing the E and NS1 proteins of JEV (rNDV-Ejev and rNDV-NS1jev). The recovered rNDV-Ejev and rNDV-NS1jev were characterized in 9-day-old SPF embryonated chicken eggs and in cell culture. The vaccination of rNDV-Ejev and rNDV-NS1jev showed effective immunity against JEV upon intranasal immunization in BALB/c mice. The rNDVs vaccination produced effective neutralization antibody titers against both NDV and JEV. The cytokine profiling of the vaccinated mice showed an effective Th1 and Th2 mediated immune response. The study also provided an insight that E, when used in combination with NS1 could reduce the efficacy of only E based immunization in mice. Our results suggested rNDV-Ejev to be a promising live viral vectored vaccine against JEV. This study implies an alternative and economical strategy for the development of a recombinant vaccine against JEV.     

Japanese encephalitis virus vaccine candidates generated by chimerization with dengue virus type 4

Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis worldwide and vaccination is one of the most effective ways to prevent disease. A suitable live-attenuated JEV vaccine could be formulated with a live-attenuated tetravalent dengue vaccine for the control of these viruses in endemic areas. Toward this goal, we generated chimeric virus vaccine candidates by replacing the precursor membrane (prM) and envelope (E) protein structural genes of recombinant dengue virus type 4 (rDEN4) or attenuated vaccine candidate rDEN4Δ30 with those of wild-type JEV strain India/78. Mutations were engineered in E, NS3 and NS4B protein genes to improve replication in Vero cells. The chimeric viruses were attenuated in mice and some elicited modest but protective levels of immunity after a single dose. One particular chimeric virus, bearing E protein mutation Q264H, replicated to higher titer in tissue culture and was significantly more immunogenic in mice. The results are compared with live-attenuated JEV vaccine strain SA14-14-2.     

Intranasal VLP-RBD vaccine adjuvanted with BECC470 confers immunity against Delta SARS-CoV-2 challenge in K18-hACE2-mice

As the COVID-19 pandemic transitions into endemicity, seasonal boosters are a plausible reality across the globe. We hypothesize that intranasal vaccines can provide better protection against asymptomatic infections and more transmissible variants of SARS-CoV-2. To formulate a protective intranasal vaccine, we utilized a VLP-based platform. Hepatitis B surface antigen-based virus like particles (VLP) linked with receptor binding domain (RBD) antigen were paired with the TLR4-based agonist adjuvant, BECC 470. K18-hACE2 mice were primed and boosted at four-week intervals with either VLP-RBD-BECC or mRNA-1273. Both VLP-RBD-BECC and mRNA-1273 vaccination resulted in production of RBD-specific IgA antibodies in serum. RBD-specific IgA was also detected in the nasal wash and lung supernatants and were highest in VLP-RBD-BECC vaccinated mice. Interestingly, VLP-RBD-BECC vaccinated mice showed slightly lower levels of pre-challenge IgG responses, decreased RBD-ACE2 binding inhibition, and lower neutralizing activity in vitro than mRNA-1273 vaccinated mice. Both VLP-RBD-BECC and mRNA-1273 vaccinated mice were protected against challenge with a lethal dose of Delta variant SARS-CoV-2. Both vaccines limited viral replication and viral RNA burden in the lungs of mice. CXCL10 is a biomarker of severe SARS-CoV-2 infection and we observed both vaccines limited expression of serum and lung CXCL10. Strikingly, VLP-RBD-BECC when administered intranasally, limited lung inflammation at early timepoints that mRNA-1273 vaccination did not. VLP-RBD-BECC immunization elicited antibodies that do recognize SARS-CoV-2 Omicron variant. However, VLP-RBD-BECC immunized mice were protected from Omicron challenge with low viral burden. Conversely, mRNA-1273 immunized mice had low to no detectable virus in the lungs at day 2. Together, these data suggest that VLP-based vaccines paired with BECC adjuvant can be used to induce protective mucosal and systemic responses against SARS-CoV-2.     

Antibody persistence of two pentavalent DTwP–HB–Hib vaccines to the age of 15–18 months,and response to the booster dose of quadrivalent DTwP–Hib vaccine

Objectives

Antibody persistence in children following three doses of primary vaccination with diphtheria, tetanus, whole-cell-pertussis (DTwP), hepatitis B, and Haemophilus influenzae type b (Hib) vaccines (SIIL Pentavac vaccine vs. Easyfive^® of Panacea Biotec), and response to the booster dose of DTwP–Hib (Quadrovax^®) vaccine.

Methods

Children who completed their primary immunization were assessed for antibodies at 15–18 months of age, and then given a booster dose of DTwP–Hib vaccine. Reactogenicity and safety of the booster dose was evaluated.

Results

Both pentavalent vaccines demonstrated a good immune response at 15–18 months. Following the booster dose, all vaccinated subjects achieved protective titers against diphtheria, tetanus and Hib, whereas the response to pertussis antigen was ∼78%. Fever and irritability was noted in 24%, local pain in 51%, and swelling in 36% of the children following booster dose.

Conclusions

Primary immunization with either pentavalent vaccine induced an excellent immunity lasting till the second year of life. A booster dose with DTwP–Hib (Quadrovax^®) vaccine effectuated a good anamnestic response to all vaccine components, being specially strong for Hib in children previously vaccinated with SIIL liquid pentavalent vaccine (Pentavac^®). Also, the safety profile of SIIL quadrivalent vaccine (Quadrovax^®) administered as booster dose was acceptable.     

Minimal immune response to booster vaccination against Yellow Fever associated with pre-existing antibodies

Ever since its development in the 1930′s, the live-attenuated Yellow Fever virus vaccine YF-17D has been highly effective. Despite the increasing knowledge on the immune biology of the YF-17D vaccine, most studies have focused only on a few types of immune cells and pathways or mainly on the primary adaptive immune response to YF-17D vaccination. Here, we examined humoral, innate and adaptive cellular responses in a longitudinal YF-17D vaccination study in Switzerland, comparing both primary and booster vaccination. In contrast to the strong innate and adaptive immune response to the primary vaccination, we find that the response to boosting is much reduced. Our data show an inverse association of neutralizing antibodies at baseline with vaccine virus replication and with the immune response upon boosting. These results suggest that booster vaccination may not have major immunological effects when neutralizing antibodies are present. Importantly, our study population was healthy adults in a non-endemic country and ultimately booster vaccine requirement must be assessed based on additional epidemiological and public health considerations in endemic areas.     

Colostral antibody protection and interference with immunity in lambs born from sheep vaccinated with an inactivated Bluetongue serotype 8 vaccine

Widespread vaccination programmes against Bluetongue virus serotype 8 (BTV-8), using inactivated vaccines, are being carried out across many countries in northern, western and southern Europe. This study investigates the extent and length of colostral antibody protection, as well as the degree of colostral antibody induced interference of the immune response to BTV-8, in sheep. Significantly lower titres of neutralising antibodies were transferred in colostrum to lambs born from sheep vaccinated once as opposed those vaccinated twice (single vaccine in the first year and a booster vaccine in the second year). On BTV-8 challenge, lambs born from sheep vaccinated on two occasions, with the second booster vaccine given approximately 1 month prior to lambing, were protected from clinical disease for up to 14 weeks. BTV-8 was isolated from 5 of the 22 challenged lambs, although only one of these lambs showed a transient rise in body temperature with no other clinical signs. Lambs born from ewes given a second booster vaccine 1 month prior to lambing, are likely to be protected from clinical disease for at least 14 weeks, whereas lambs born from ewes vaccinated once are likely to be protected for a shorter time. Colostral antibodies present in the 13–14-week-old lambs appeared to interfere with the humoral response to challenge virus. These results suggest that colostral antibodies may interfere with vaccination in lambs up to at least 14 weeks of age.     

A recombinant virus vaccine that protects against both Chikungunya and Zika virus infections

Chikungunya virus (CHIKV) and Zika virus (ZIKV) have recently expanded their range in the world and caused serious and widespread outbreaks of near pandemic proportions. There are no licensed vaccines that protect against these co-circulating viruses that are transmitted by invasive mosquito vectors. We report here on the development of a single-dose, bivalent experimental vaccine for CHIKV and ZIKV. This vaccine is based on a chimeric vesicular stomatitis virus (VSV) that expresses the CHIKV envelope polyprotein (E3-E2-6K-E1) in place of the VSV glycoprotein (G) and also expresses the membrane-envelope (ME) glycoproteins of ZIKV. This vaccine induced neutralizing antibody responses to both CHIKV and ZIKV in wild-type mice and in interferon receptor-deficient A129 mice, animal models for CHIKV and ZIKV infection. A single vaccination of A129 mice with the vector protected these mice against infection with both CHIKV and ZIKV. Our single-dose vaccine could provide durable, low-cost protection against both CHIKV and ZIKV for people traveling to or living in areas where both viruses are circulating, which include most tropical regions in the world.