Nipah virus (NiV) is a highly pathogenic re-emerging virus that causes outbreaks in South East Asia. Currently, no approved and licensed vaccine or antivirals exist. Here, we investigated the efficacy of ChAdOx1 NiVB, a simian adenovirus-based vaccine encoding NiV glycoprotein (G) Bangladesh, in Syrian hamsters. Prime-only as well as prime-boost vaccination resulted in uniform protection against a lethal challenge with NiV Bangladesh: all animals survived challenge and we were unable to find infectious virus either in oral swabs, lung or brain tissue. Furthermore, no pathological lung damage was observed. A single-dose of ChAdOx1 NiVB also prevented disease and lethality from heterologous challenge with NiV Malaysia. While we were unable to detect infectious virus in swabs or tissue of animals challenged with the heterologous strain, a very limited amount of viral RNA could be found in lung tissue by in situ hybridization. A single dose of ChAdOx1 NiVB also provided partial protection against Hendra virus and passive transfer of antibodies elicited by ChAdOx1 NiVB vaccination partially protected Syrian hamsters against NiV Bangladesh. From these data, we conclude that ChAdOx1 NiVB is a suitable candidate for further NiV vaccine pre-clinical development.
The major outer membrane protein (OmpH) of 4 local Malaysian strains of Pasteurella multocida serotype B:2 were characterized in comparison to ATCC strains. Three major peptide bands of MW 26, 32 and 37 kDa were characterized using SDSPAGE. Two of these fragments, the 32 kDa and 37 kDa were observed to be more reactive with a mouse polyclonal antiserum in all of the local isolates as well as the ATCC strains in a Western blot. However, the 32 kDa fragment was found to cross react with other Gram negative bacteria. Therefore, the 37 kDa OmpH was selected as vaccine candidate. The 37 kDa ompH gene of the isolated strain 1710 was cloned into an Escherichia coli expression vector to produce large amounts of recombinant OmpH (rOmpH). The 37 kDa ompH gene of strain 1710 was sequenced. In comparison to a reference strain X-73 of the ompH of P. multocida, 39bp was found deleted in the 37 kDa ompH gene. However, the deletion did not shift the reading frame or change the amino acid sequence. The rOmpH was used in a mice protection study. Mice immunized and challenged intraperitoneally resulted 100% protection against P. multocida whilst mice immunized subcutaneously and challenged intraperitoneally only resulted 80% protection. The rOmpH is therefore a suitable candidate for vaccination field studies. The same rOmpH was also used to develop a potential diagnostic kit in an ELISA format.
Shigellosis is one of the most common diseases found in the developing countries, especially those countries that are prone flood. The causative agent for this disease is the Shigella species. This organism is one of the third most common enteropathogens responsible for childhood diarrhea. Since Shigella can survive gastric acidity and is an intracellular pathogen, it becomes difficult to treat. Also, uncontrolled use of antibiotics has led to development of resistant strains which poses a threat to public health. Therefore, there is a need for long term control of Shigella infection which can be achieved by designing a proper and effective vaccine. In this study, emphasis was made on designing a candidate that could elicit both B-cell and T-cell immune response. Hence B- and T-cell epitopes of outer membrane channel protein (OM) and putative lipoprotein (PL) from S. flexneri 2a were computationally predicted using immunoinformatics approach and a chimeric construct (chimeric-OP) containing the immunogenic epitopes selected from OM and PL was designed, cloned and expressed in E. coli system. The immunogenicity of the recombinant chimeric-OP was assessed using Shigella antigen infected rabbit antibody. The result showed that the chimeric-OP was a synthetic peptide candidate suitable for the development of vaccine and immunodiagnostics against Shigella infection.
Human respiratory syncytial virus is the most common cause of bronchiolitis and other respiratory infections in infants and the elderly worldwide. We have developed two new oral vaccines using Salmonella typhi TY21a to carry and express the immunogenic epitopes of RSV fusion (F) and attachment (G) glycoproteins on its surface, separately. To evaluate the efficacy of the designed vaccines, BALB/c mice were orally immunized and then infected with RSV. Immune response analyses showed that cellmediated, mucosal and humoral immunity in the vaccinated mice were significantly enhanced compared to the control group. Both vaccines generated a balanced Th1/Th2 immune response which is crucial for efficiency of vaccines against RSV. Furthermore, histopathological examination proved that these vaccines were safe as they did not cause any Th2-associated adverse effects in the lungs of RSV-infected mice. The findings of this research suggest that Salmonella-F and Salmonella-G vaccine candidates may have strong potential to prevent RSV infection.
The ability of a heat-inactivated whole virus from a highly virulent infectious bursal disease virus (hvIBDV) and VP2 protein from hvIBDV expressed in E. coli provided protection against a hvIBDV challenge in specificpathogen-free (SPF) chickens. Six out of seven chickens that were injected three times with crude VP2 protein developed significant antibody titer against IBDV. However, only four out of the seven chickens survived the hvIBDV challenge. Despite showing low antibody titer profiles, all chickens immunized with the heat-inactivated whole virus also survived the challenged with hvIBDV. However, all of these chickens had bursal atrophy and mild to moderate depletion of lymphocytes. Thus, antibodies raised against IBDV VP2 protein expressed in E. coli and denatured IBDV proteins induced some degree of protection against mortality but not against bursal damage following challenge with hvIBDV.
Toxoplasmosis, a parasitic disease in human and animals, is caused by Toxoplasma gondii. Our previous study has led to the discovery of a novel RAP domain binding protein antigen (TgRA15), an apparent in-vivo induced antigen recognised by antibodies in acutely infected individuals. This study is aimed to evaluate the humoral response and cytokine release elicited by recombinant TgRA15 protein in C57BL/6 mice, demonstrating its potential as a candidate vaccine for Toxoplasma gondii infection. In this study, the recombinant TgRA15 protein was expressed in Escherichia coli, purified and refolded into soluble form. C57BL/6 mice were immunised intradermally with the antigen and CASAC (Combined Adjuvant for Synergistic Activation of Cellular immunity). Antigen-specific humoral and cell-mediated responses were evaluated using Western blot and ELISA. The total IgG, IgG1 and IgG2a antibodies specific to the antigen were significantly increased in treatment group compare to control group. A higher level of interferon gamma (IFN-γ) secretion was demonstrated in the mice group receiving booster doses of rTgRA15 protein, suggesting a potential Th1-mediated response. In conclusion, the rTgRA15 protein has the potential to generate specific antibody response and elicit cellular response, thus potentially serve as a vaccine candidate against T. gondii infection.
Nipah virus (NiV) is a re-emerging zoonotic pathogen that causes high mortality in humans and pigs. Oral immunization in free-roaming animals is one of the most practical approaches to prevent NiV pandemics. We previously generated a recombinant rabies viruses (RABV) Evelyn-Rokitnicki-Abelseth (ERA) strain, rERAG333E, which contains a mutation from arginine to glutamic acid at residue 333 of glycoprotein (G333E) and serves as an oral vaccine for dog rabies. In this study, we generated two recombinant RABVs, rERAG333E/NiVG and rERAG333E/NiVF, expressing the NiV Malaysian strain attachment glycoprotein (NiV-G) or fusion glycoprotein (NiV-F) gene based on the rERAG333E vector platform. Both rERAG333E/NiVG and rERAG333E/NiVF displayed growth properties similar to those of rERAG333E and caused marked syncytia formation after co-infection in BSR cell culture. Adult and suckling mice intracerebrally inoculated with the recombinant RABVs showed NiV-G and NiV-F expression did not increase the virulence of rERAG333E. Oral vaccination with rERAG333E/NiVG either singularly or combined with rERAG333E/NiVF induced significant NiV neutralizing antibody against NiV and RABV, and IgG to NiV-G or NiV-F in mice and pigs. rERAG333E/NiVG and rERAG333E/NiVF thus appeared to be suitable candidates for further oral vaccines for potential animal targets in endemic areas of NiV disease and rabies.
Enterovirus 71 (EV71) causes severe neurological diseases resulting in high mortality in young children worldwide. Development of an effective vaccine against EV71 infection is hampered by the lack of appropriate animal models for efficacy testing of candidate vaccines. Previously, we have successfully tested the immunogenicity and protectiveness of a candidate EV71 vaccine, containing recombinant Newcastle disease virus capsids that display an EV71 VP1 fragment (NPt-VP11-100) protein, in a mouse model of EV71 infection. A drawback of this system is its limited window of EV71 susceptibility period, 2 weeks after birth, leading to restricted options in the evaluation of optimal dosing regimens. To address this issue, we have assessed the NPt-VP11-100 candidate vaccine in a hamster system, which offers a 4-week susceptibility period to EV71 infection. Results obtained showed that the NPt-VP11-100 candidate vaccine stimulated excellent humoral immune response in the hamsters. Despite the high level of antibody production, they failed to neutralize EV71 viruses or protect vaccinated hamsters in viral challenge studies. Nevertheless, these findings have contributed towards a better understanding of the NPt-VP11-100 recombinant protein as a candidate vaccine in an alternative animal model system.
Hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a vital role in the viral infectivity, host immunity, and disease diagnosis. A portion of the HN gene encoding the ectodomain (nt 142-1739) was cloned and expressed in Escherichia coli yielding an insoluble HN protein and a soluble NusA-HN protein containing N-utilization substance A (NusA) fusion component. Both recombinant proteins were purified and used for immunization of chickens. The recombinant HN protein induced higher antibody titers as compared to the recombinant NusA-HN protein. These antibodies were able to react in immunoblot analysis with the corresponding recombinant proteins as well as with the HN protein of NDV.
Burkholderia pseudomallei is resistant to a diverse group of antimicrobials including third generation cephalosporins whilst quinolones and aminoglycosides have no reliable effect. As therapeutic options are limited, development of more effective forms of immunotherapy is vital to avoid a fatal outcome. In an earlier study, we reported on the B. pseudomallei serine MprA protease, which is relatively stable over a wide pH and temperature range and digests physiological proteins. The present study was carried out to evaluate the immunogenicity and protective efficacy of the MprA as a potential vaccine candidate. In BALB/c mice immunized with recombinant MprA protease (smBpF4), a significantly high IgG titer was detectable. Isotyping studies revealed that the smBpF4-specific antibodies produced were predominantly IgG(1), proposing that immunization with smBpF4 triggered a Th2 immune response. Mice were immunized with smBpF4 and subsequently challenged with B. pseudomallei via the intraperitoneal route. Whilst control mice succumbed to the infection by day 9, smBpF4-immunized mice were protected against the lethal challenge and survived beyond 25 days post-infection. In conclusion, MprA is immunogenic in melioidosis patients whilst also eliciting a strong immune response upon bacterial challenge in mice and presents itself as a potential vaccine candidate for the treatment of melioidosis.
Recombinant Mycobacterium bovis bacille Calmette-Guèrin (rBCG) expressing the malarial epitopes F2R(II)EBA and (NANP)3 as well as two T cell epitopes of the M. tuberculosis ESAT-6 antigen, generated in favour of mycobacterium codon usage elicited specific immune response against these epitopes. Immunised Balb/c mice demonstrated an increase in almost all of the IgG subclasses against both malarial epitopes and enhanced splenocyte proliferative response against the malarial epitopes as well as selected peptides of ESAT-6. Furthermore, flow cytometric analyses showed elevated numbers of CD4+ lymphocytes expressing IFN-gamma and IL-2 against the ESAT-6 peptides, suggesting a specific Th1-mediated response. This study demonstrated that expressing malarial and TB epitopes in a single rBCG construct induced appropriate humoral and cellular immune response against immunogenic epitopes from both organisms.
The Plasmodium falciparum serine repeat antigen (SERA) is one of the promising blood-stage malarial vaccine candidates. In this study, recombinant Mycobacterium bovis bacille Calmette-Guerin (rBCG) expressing the 22 kDa protein (SE22) from the 47 kDa Nterminal domain of serine repeat antigen (SERA), generated in favour of mycobacterium codon usage, elicited specific immune response in BALB/c mice with a mixed Th1/Th2 profile. Immunized sera containing high levels of specific IgG1 and IgG2a against the epitope (as determined by ELISA) were reactive with fixed P. falciparum merozoites as demonstrated by indirect immunofluorescence assay (IFA). Furthermore, the lymphocyte proliferative response to SE22 antigen from rBCG-immunized mice was higher than that of controls. The expression of intracellular cytokines (IL-2, IL-4 and IFNγ) in CD4+- and CD8+-cells was also enhanced following in-vitro stimulation with SE22. These findings indicate that a rBCG-based vaccine candidate expressing a blood-stage antigen of P. falciparum could enhance both humoral and cellular immune responses, thus paving the way for the rational use of rBCG as a vaccine candidate against malaria.
Recombinant Mycobacterium bovis bacille Calmette-Guèrin (rBCG) expressing three T cell epitopes of Mycobacterium tuberculosis (MTB) Ag85B antigen (P1, P2, P3) fused to the Mtb8.4 protein (rBCG018) or a combination of these antigens fused to B cell epitopes from ESAT-6, CFP-10 and MTP40 proteins (rBCG032) were used to immunize Balb/c mice. Total IgG responses were determined against Mtb8.4 antigen and ESAT-6 and CFP-10 B cell epitopes after immunization with rBCG032. Mice immunized with rBCG032 showed a significant increase in IgG1 and IgG2a antibodies against ESAT-6 and MTP40 (P1) B cell epitopes and IgG3 against both P1 and P2 B cell epitopes of MPT40. Splenocytes from mice immunized with rBCG018 proliferated against Ag85B P2 and P3 T cell epitopes and Mtb8.4 protein whereas those from mice-immunized with rBCG032 responded against all Ag85B epitopes and the ESAT-6 B cell epitope. CD4⁺ and CD8⁺ lymphocytes from mice immunized with rBCG018 produced primarily Th1 type cytokines in response to the T cell epitopes. Similar pattern of recognition against the T cell epitopes were obtained with rBCG032 with the additional recognition of ESAT-6, CFP-10 and one of the MTP40 B cell epitopes with the same pattern of cytokines. This study demonstrates that rBCG constructs expressing either T or T and B cell epitopes of MTB induced appropriate immunogenicity against MTB.
Hepatitis B virus (HBV) is a deadly pathogen that has killed countless people worldwide. Saccharomyces cerevisiae-derived HBV vaccines based upon hepatitis B surface antigen (HBsAg) is highly effective. However, the emergence of vaccine escape mutants due to mutations on the HBsAg and polymerase genes has produced a continuous need for the development of new HBV vaccines. In this study, the "a" determinant within HBsAg was displayed on the recombinant capsid protein of Macrobrachium rosenbergii nodavirus (MrNV), which can be purified easily in a single step through immobilized metal affinity chromatography (IMAC). The purified protein self-assembled into virus-like particles (VLPs) when observed under a transmission electron microscope (TEM). Immunization of BALB/c mice with this chimeric protein induced specific antibodies against the "a" determinant. In addition, it induced significantly more natural killer and cytotoxic T cells, as well as an increase in interferon gamma (IFN-γ) secretion, which are vital for virus clearance. Collectively, these findings demonstrated that the MrNV capsid protein is a potential carrier for the HBV "a" determinant, which can be further extended to display other foreign epitopes. This paper is the first to report the application of MrNV VLPs as a novel platform to display foreign epitopes.
To evaluate a live recombinant Lactococcus lactis vaccine expressing aerolysin genes D1 (Lac-D1ae) and/or D4 (Lac-D4ae) in protection against Aeromonas hydrophila in tilapia (Oreochromis niloticus).
A gene associated with lipopolysaccharide (LPS) transport was cloned from a local clinical Vibrio cholerae O1 strain of the Ogawa serotype by using the Lactococcus lactis nisin-controlled expression (NICE) system. The V. cholerae wzm gene, which codes for an integral membrane transporter protein, was expressed and targeted to the cytoplasmic membrane, and was crudely isolated through simple centrifugation and SDS solubilization. To examine seroreactivity of this construct, rabbits were orally fed with 10(9) cfu/ml of live, recombinant L. lactis carrying the wzm gene, induced with nisin prior to administration. Recombinant plasmids were retrieved from L. lactis cultured directly from stool samples of inoculated rabbits. Reverse-transcriptase PCR of wzm using the retrieved plasmids confirmed transcription of this gene, indicating viability and stability of the recombinants in vivo. The L. lactis-Wzm construct elicited substantial levels of IgG and sIgA, and challenge with virulent V. cholerae O1 evoked severe diarrhoea in the naive, non-immunised control group, but not in those fed with either recombinant or non-recombinant L. lactis. Oral administration with recombinant L. lactis expressing the V. cholerae wzm gene increases both systemic and mucosal immunity, whereas L. lactis itself appears capable of protecting against the diarrhoeal symptoms caused by V. cholerae. Wzm is a conserved membrane protein associated with the LPS endotoxin, and together with the food-grade L. lactis, represent an attractive target for the development of a safer, live anti-infective therapy against V. cholerae.
Enterovirus 71 (EV71) infection may cause severe neurological complications, particularly in young children. Despite the risks, there are still no commercially available EV71 vaccines. Hence, a candidate vaccine construct, containing recombinant Newcastle disease virus capsids that display an EV71 VP1 fragment (NPt-VP1(1-100) ) protein, was evaluated in a mouse model of EV71 infection. Previously, it was shown that this protein construct provoked a strong immune response in vaccinated adult rabbits. That study, however, did not address the issue of its effectiveness against EV71 infection in young animals. In the present study, EV71 viral challenge in vaccinated newborn mice resulted in more than 40% increase in survival rate. Significantly, half of the surviving mice fully recovered from their paralysis. Histological analysis of all of the surviving mice revealed a complete clearance of EV71 viral antigens from their brains and spinal cords. In hind limb muscles, the amounts of the antigens detected correlated with the degrees of tissue damage and paralysis. Findings from this study provide evidence that immunization with the NPt-VP1(1-100) immunogen in a newborn mouse model confers partial protection against EV71 infection, and also highlights the importance of NPt-VP1(1-100) as a possible candidate vaccine for protection against EV71 infections.
Highly pathogenic avian influenza (HPAI) H5N1 is an ongoing global health concern due to its severe sporadic outbreaks in Asia, Africa and Europe, which poses a potential pandemic threat. The development of safe and cost-effective vaccine candidates for HPAI is considered the best strategy for managing the disease and addressing the pandemic preparedness. The most potential vaccine candidate is the antigenic determinant of influenza A virus, hemagglutinin (HA). The present research was aimed at developing optimized expression in Nicotiana benthamiana and protein purification process for HA from the Malaysian isolate of H5N1 as a vaccine antigen for HPAI H5N1. Expression of HA from the Malaysian isolate of HPAI in N. benthamiana was confirmed, and more soluble protein was expressed as truncated HA, the HA1 domain over the entire ectodomain of HA. Two different purification processes were evaluated for efficiency in terms of purity and yield. Due to the reduced yield, protein degradation and length of the 3-column purification process, the 2-column method was chosen for target purification. Purified HA1 was found immunogenic in mice inducing H5 HA-specific IgG and a hemagglutination inhibition antibody. This paper offers an alternative production system of a vaccine candidate against a locally circulating HPAI, which has a regional significance.
Nipah virus (NiV), of the family Paramyxoviridae, was isolated in 1999 in Malaysia from a human fatality in an outbreak of severe human encephalitis, when human infections were linked to transmission of the virus from pigs. Consequently, a swine vaccine able to abolish virus shedding is of veterinary and human health interest. Canarypox virus-based vaccine vectors carrying the gene for NiV glycoprotein (ALVAC-G) or the fusion protein (ALVAC-F) were used to intramuscularly immunize four pigs per group, either with 10(8) PFU each or in combination. Pigs were boosted 14 days postvaccination and challenged with 2.5 x 10(5) PFU of NiV two weeks later. The combined ALVAC-F/G vaccine induced the highest levels of neutralization antibodies (2,560); despite the low neutralizing antibody levels in the F vaccinees (160), all vaccinated animals appeared to be protected against challenge. Virus was not isolated from the tissues of any of the vaccinated pigs postchallenge, and a real-time reverse transcription (RT)-PCR assay detected only small amounts of viral RNA in several samples. In challenge control pigs, virus was isolated from a number of tissues (10(4.4) PFU/g) or detected by real-time RT-PCR. Vaccination of the ALVAC-F/G vaccinees appeared to stimulate both type 1 and type 2 cytokine responses. Histopathological findings indicated that there was no enhancement of lesions in the vaccinees. No virus shedding was detected in vaccinated animals, in contrast to challenge control pigs, from which virus was isolated from the throat and nose (10(2.9) PFU/ml). Based on the data presented, the combined ALVAC-F/G vaccine appears to be a very promising vaccine candidate for swine.