Assessing the protection elicited by virus-like particles expressing the RSV pre-fusion F and tandem repeated G proteins against RSV rA2 line19F infection in mice.

Excessive pulmonary inflammation is the hallmark of respiratory syncytial virus (RSV) infection hindering efficacious RSV vaccine development. Yet, the vast majority of the experimental RSV vaccine studies use laboratory-adapted RSV strains that do not reflect the highly pathogenic and inflammatory nature of the virus found in clinical settings. Here, we re-evaluated the protective efficacy of the virus-like particle (VLP) vaccine co-expressing the pre-fusion (pre-F) protein and G protein with tandem repeats (Gt) reported in our previous study against the recombinant RSV rA2-line19F strain, which inflicts severe mucus production and inflammation in mice. VLP vaccine immunization elicited virus-specific serum antibody responses that mediated RSV rA2-line19F virus neutralization. VLP vaccine immunization promoted Th1 immune response development in the spleens and CD8 + T cell influx into the lungs of mice, which are essential for efficient viral clearance and dampened inflammatory response. When compared to the VLPs expressing only the pre-F antigen, those co-expressing both pre-F and Gt antigens conferred better protection in mice against rA2-line19F challenge infection. Overall, our data suggest that the pre-clinical VLP vaccine co-expressing RSV pre-F and Gt antigens can effectively protect mice against RSV strains that resemble pathogenic clinical isolates.

Influenza virus-like particle vaccine containing both apical membrane antigen 1 and microneme-associated antigen proteins of Plasmodium berghei confers protection in mice.

BACKGROUND: Apical membrane antigen 1 (AMA1) and microneme-associated antigen (MIC) of Plasmodium parasites are important factors involved in host cell invasion. METHODS: In this study, influenza VLP vaccines containing both codon-optimized AMA1 and MIC were generated and the vaccine efficacy was evaluated in mice. RESULTS: VLPs vaccine immunization elicited higher levels of parasite-specific IgG and IgG2a antibody responses in sera. CD4+ and CD8+ T cells and germinal center B cells in blood, inguinal lymph nodes (ILN) and spleen were found to be significantly increased. Importantly, VLPs vaccination significantly reduced the levels of pro-inflammatory cytokines IFN-γ and TNF-α, decreased parasitemia in blood, resulting in lower body weight loss and longer survival time compared to control. CONCLUSION: These results indicated that VLPs containing P. berghei AMA1 and MIC could be a candidate for malaria blood-stage vaccine design.

Virus-Like Particle Vaccines Against Respiratory Viruses and Protozoan Parasites.

The field of vaccinology underwent massive advances over the past decades with the introduction of virus-like particles (VLPs), a supra-molecular nanoparticle vaccine platform that resembles viral structures without the ability to replicate in hosts. This innovative approach has been remarkably effective, as evidenced by its profound immunogenicity and safety. These highly desirable intrinsic properties enabled their further development as vaccines against a multitude of diseases. To date, several VLP-based vaccines have already been commercialized and many more are undergoing clinical evaluation prior to FDA approval. However, efficacious vaccines against a plethora of pathogens are still lacking, which imposes a tremendous socioeconomic burden and continues to threaten public health throughout the globe. This is especially the case for several respiratory pathogens and protozoan parasites. In this review, we briefly describe the fundamentals of VLP vaccines and the unique properties that enable these to be such valuable vaccine candidates and summarize current advances in VLP-based vaccines targeting respiratory and parasitic diseases of global importance.

Protective immunity induced by CpG ODN-adjuvanted virus-like particles containing Toxoplasma gondii proteins.

AIMS: To date, a Toxoplasma gondii vaccine for clinical use remains unavailable, though multiple vaccine candidates have been suggested. In our previous studies, unadjuvanted virus-like particles (VLPs) vaccines expressing multiple T. gondii antigens were confirmed to be protective against T. gondii challenge infection. Yet, the protective efficacy of adjuvanted T. gondii VLP in comparison with the unadjuvanted counterpart requires elucidation. METHODS AND RESULTS: In the present study, mice were immunized with the multi-antigenic VLP vaccines (TG146 VLP) with or without CpG adjuvants and their protective efficacies were compared. CpG-adjuvanted TG146 VLP vaccine elicited enhanced T gondii-specific IgG and IgA antibody responses in the sera, mucosal tissue and the brain compared to unadjuvanted VLPs vaccine. Inclusion of CpG adjuvant in vaccines also induced greater CD4+ and CD8+ T-cell responses, as well as B cell and germinal centre B cell responses from splenocytes and mesenteric lymph nodes. Pro-inflammatory cytokine response and cyst counts in the brain were drastically diminished in mice immunized with CpG-adjuvanted VLP vaccines. CONCLUSION: Our results demonstrated that CpG-adjuvanted T. gondii VLPs can significantly enhance the protective efficacy of vaccines against T. gondii infection.

Detection of Toxoplasma gondii Infections using Virus-Like Particles Displaying T. gondii ROP4 Antigen.

Toxoplasma gondii ME49 infections are typically diagnosed by serological tests. However, serological diagnosis of RH strain-induced toxoplasmosis remains unknown. In order to develop seradiagnosis of above 2 kinds of infections, we generated recombinant virus-like particles (VLPs) displaying the T. gondii rhoptry protein 4 (ROP4) and evaluated their potential in T. gondii ME49 or RH strain infection diagnostics. Mice were orally infected with either the tachyzoites of T. gondii (RH) or cysts of T. gondii (ME49) at various dosages, and sera were collected at regular intervals. ELISA-based serological tests were performed to assess IgG, IgM, and IgA antibody responses against ROP4 VLP antigen and tissue lysate antigen (TLA). Compared to TLA, IgG, IgM, and IgA levels to ROP4 VLP antigen were significantly higher in the sera of T. gondii RH-infected mice 1 and 2 week post-infection (PI). T. gondii-specific IgG antibody was detected at 1, 2, 4, and 8 week PI in the T. gondii ME49-infected mice with infection dose-dependent manner. These results indicated that the ROP4 VLP antigen was highly sensitive antigens detecting T. gondii RH and ME49 antibodies at an early stage.

Differentially Expressed Gene Profile of Acanthamoeba castellanii Induced by an Endosymbiont Legionella pneumophila.

Legionella pneumophila is an opportunistic pathogen that survives and proliferates within protists such as Acanthamoeba spp. in environment. However, intracellular pathogenic endosymbiosis and its implications within Acanthamoeba spp. remain poorly understood. In this study, RNA sequencing analysis was used to investigate transcriptional changes in A. castellanii in response to L. pneumophila infection. Based on RNA sequencing data, we identified 1,211 upregulated genes and 1,131 downregulated genes in A. castellanii infected with L. pneumophila for 12 hr. After 24 hr, 1,321 upregulated genes and 1,379 downregulated genes were identified. Gene ontology (GO) analysis revealed that L. pneumophila endosymbiosis enhanced hydrolase activity, catalytic activity, and DNA binding while reducing oxidoreductase activity in the molecular function (MF) domain. In particular, multiple genes associated with the GO term 'integral component of membrane' were downregulated during endosymbiosis. The endosymbiont also induced differential expression of various methyltransferases and acetyltransferases in A. castellanii. Findings herein are may significantly contribute to understanding endosymbiosis of L. pneumophila within A. castellanii.

Application of Histone Deacetylase Inhibitors MPK472 and KSK64 as a Potential Treatment Option for Acanthamoeba Keratitis.

Treatment of Acanthamoeba keratitis (AK) is difficult because Acanthamoeba cysts are resistant to drugs, and as such, successful treatment requires an effective approach that inhibits cyst formation. Histone deacetylase inhibitors (HDACis) are involved in cell proliferation, differentiation, and apoptotic cell death. In this study, the effects of HDACis such as MPK472 and KSK64 on Acanthamoeba castellanii trophozoites and cysts were observed. MPK472 and KSK64 showed at least 60% amoebicidal activity against Acanthamoeba trophozoites at a concentration of 10 µM upon 8 h of treatment. Neither of the two HDACis affected mature cysts, but significant amoebicidal activities (36.4 and 33.9%) were observed against encysting Acanthamoeba following treatment with 5 and 10 µM HDACis for 24 h. Light microscopy and transmission electron microscopy results confirmed that the encystation of Acanthamoeba was inhibited by the two HDACis. In addition to this, low cytopathic effects on human corneal epithelial (HCE) cells were observed following treatment with MPK472 and KSK64 for 24 h. Our results indicate that the HDACis MPK472 and KSK64 could be used as new candidates for the development of an optimal therapeutic option for AK.

Orally administered recombinant baculovirus vaccine elicits partial protection against avian influenza virus infection in mice.

Avian influenza outbreaks have placed a tremendous economic burden on the poultry industry, necessitating the need for an effective vaccine. Although multiple vaccine candidates are available, its development is hindered by several drawbacks associated with the vaccine platforms and as such, more improvements to the vaccines are needed. Therefore, in this study, the vaccine efficacy in the murine models was assessed prior to evaluation in chickens. An oral recombinant baculovirus (rBV) vaccine expressing influenza hemagglutinin (HA) (A/H5N1) was generated and its efficacy was investigated against homologous avian influenza infection in mice. Our results confirmed that oral administration of rBVs enhanced the level of virus-specific antibodies in the sera following boost immunization. Upon challenge infection with a lethal dose of highly pathogenic avian influenza virus (HPAI, H5N1) virus, a marked increase in mucosal IgG and IgA were observed. Drastically increased antibody secretory cell responses from the bone marrow cells and splenocytes of vaccinated mice were observed, in addition to the strongly elicited germinal center responses in the lungs and the spleens. Vaccinated mice showed significantly reduced lung pro-inflammatory cytokine responses, lung viral loads, body weight loss, and mortality. Though mice were only partially protected upon challenge infection, these results highlight the potential of orally administered rBVs expressing the HA as a vaccine candidate for controlling avian influenza outbreaks.

Virus-like particle vaccine displaying Toxoplasma gondii apical membrane antigen 1 induces protection against T. gondii ME49 infection in mice.

Toxoplasmosis is an intracellular parasitic disease caused by the protozoa Toxoplasma gondii, which affects about half of the world's population. In spite of the strenuous endeavors, a T. gondii vaccine for clinical use remains unreported to date. In the present study, we generated virus-like particles (VLPs) containing T. gondii apical membrane antigen 1 (AMA1) and assessed its efficacy in a murine model. VLPs were characterized using western blot and TEM. T. gondii-specific IgG and IgA antibody responses in sera, germinal center B cell responses in spleen, brain cyst counts and their sizes were determined. Elevated T. gondii-specific IgG and IgA antibody responses were observed from the sera of AMA1 VLP-immunized mice. Immunization with AMA1 VLPs enhanced T. gondii-specific antibody-secreting cell responses and germinal center B cell responses upon antigen stimulation. Brain tissue analysis revealed that AMA1 VLP-immunization reduced cyst formation and its size compared to control. Also, VLP-immunized mice were less susceptible to body weight loss and displayed enhanced survival rate compared to the control group. Our results demonstrated that the immune response induced by T. gondii AMA1 VLPs confer partial protection against T. gondii infection and provides important insight into potential T. gondii vaccine design strategy.

Toxoplasma gondii virus-like particle vaccination alleviates inflammatory response in the brain upon T gondii infection.

AIMS: Neuroinflammation can manifest upon infection with the neurotropic parasite Toxoplasma gondii (ME49), which can lead to brain injury and cognitive dysfunction. Rhoptry organelle proteins (ROPs) secreted by T gondii play critical roles in host invasion. METHODS AND RESULTS: In this study, influenza virus-like particles (VLPs) expressing T gondii ROP4 or ROP13 were generated to assess vaccination-induced changes in intracranial pro-inflammatory cytokines and antibody responses upon T gondii challenge infection. Compared to ROP13 VLPs, ROP4VLPs vaccination significantly limited the production of pro-inflammatory cytokines IFN-γ and IL-6 in the brains of mice. Reduced pro-inflammatory cytokine responses by ROP4 VLPs and ROP13 VLPs correlated with significantly increased T gondii-specific IgG and IgA antibody responses in the brain, as well as IgG, IgG1 and IgM antibody responses in the sera. CONCLUSION: We concluded that influenza T gondii VLP vaccination induces antibody responses in sera and brain, which may contribute to the significant reduction of neuroinflammation during T gondii infection.

Virus-like particles expressing Plasmodium berghei MSP-8 induce protection against P. berghei infection.

AIMS: Merozoite surface protein 8 (MSP-8) of Plasmodium parasites plays an important role in erythrocyte invasion and is a potential malaria vaccine candidate. METHODS AND RESULTS: In this study, virus-like particles (VLPs) expressing MSP-8 of Plasmodium berghei on the surface of influenza virus matrix protein 1 (M1) core protein were generated for vaccine efficacy assessment. Mice were intramuscularly (IM) immunized with MSP-8 VLPs twice and challenge-infected with P. berghei. We found that VLP vaccination elicited higher levels of P. berghei-specific IgG antibody response in the sera, along with blood CD4+ and CD8+ T-cell response enhancement compared to the naïve control mice. CD4+ and CD8+ effector memory T-cell and memory B-cell responses in the spleen were found to be higher in VLP-immunized mice compared to control mice. VLP vaccination significantly reduced inflammatory cytokine (IFN-γ) response in the spleen and parasitemia levels in blood compared to naïve control mice. CONCLUSIONS: These results indicate that MSP-8 containing virus-like particles could be a vaccine candidate for blood-stage vaccine design.

Vaccine Efficacy Induced by 2009 Pandemic H1N1 Virus-Like Particles Differs from that Induced by Split Influenza Virus.

Influenza virus-like particles (VLPs) vaccines are highly immunogenic, showing strong protective efficacy against homologous virus infection compared to split vaccine. However, a comparative efficacy study against heterosubtypic virus infection between VLPs and split vaccine has yet to been reported. In this study, we generated VLPs vaccine containing hemagglutinin (HA) and matrix protein (M1) of the 2009 pandemic H1N1, and investigated the protective efficacies induced by VLPs vaccine and commercial monovalent H1N1 pandemic split vaccine from Sanofi-Pasteur. Mice were intramuscularly immunized with either VLPs vaccine or split vaccine and subsequently challenge-infected with homologous virus (A/California/04/2009, H1N1) or heterosubtypic virus (A/Philippines/82, H3N2) after 4.5 months. VLPs vaccination demonstrated a higher level of protective efficacy against homologous viruses compared to split vaccine, as lessened lung viral loads and minuscule levels of proinflammatory lung cytokines IFN-gamma and IL-6 were observed. Protective efficacies were close to non-existent in VLP-immunized mice challenged with heterosubtypic viruses (H3N2). In contrast, split vaccine showed lower vaccine efficacy against homologous virus than VLP vaccine, but conferred better protection against heterosubtypic viruses through lung viral loads reduction and heightened survival rate. These results indicate that influenza VLPs provide better protective efficacy against homologous virus challenge infection, whereas split vaccine shows better protective efficacy against heterosubtypic virus challenge. Findings from the current study contribute to the rational design of vaccines conferring a broad range of protection.

Protection induced by malaria virus-like particles containing codon-optimized AMA-1 of Plasmodium berghei.

BACKGROUND: Despite the extensive endeavours, developing an effective malaria vaccine remains as a great challenge. Apical membrane antigen 1 (AMA-1) located on the merozoite surface of parasites belonging to the genus Plasmodium is involved in red blood cell invasion. METHODS: Influenza virus-like particle (VLP) vaccines containing codon-optimized or native (non-codon optimized) AMA-1 from Plasmodium berghei were generated. VLP-induced protective immunity was evaluated in a mouse model. RESULTS: Mice immunized with VLP vaccine containing the codon-optimized AMA-1 elicited higher levels of P. berghei-specific IgG and IgG2a antibody responses compared to VLPs containing non-codon optimized AMA-1 before and after challenge infection. Codon-optimized AMA-1 VLP vaccination induced higher levels of CD4+ T cells, CD8+ T cells, B cells, and germinal centre cell responses compared to non-codon optimized AMA-1 VLPs. Importantly, the codon-optimized AMA-1 VLP vaccination showed lower body weight loss, longer survival and a significant decrease in parasitaemia compared to non-codon optimized VLP vaccination. CONCLUSION: Overall, VLP vaccine expressing codon-optimized AMA-1 induced better protective efficacy than VLPs expressing the non-codon optimized AMA-1. Current findings highlight the importance of codon-optimization for vaccine use and its potential involvement in future malaria vaccine design strategies.

Immune Correlates of Protection Induced by Virus-Like Particles Containing 2009 H1N1 Pandemic Influenza HA, NA or M1 Proteins.

Influenza virus-like particle (VLPs) vaccines are a promising alternative to conventional egg-based vaccines. Evaluation of vaccine efficacy induced by HA-M1 VLPs, NA-M1 VLPs or M1 VLPs against virus challenge infection would provide important insight into vaccine design strategy. In this study, we generated VLPs containing hemagglutinin (HA), neuraminidase (NA) or M1 proteins derived from the A/California/04/09. Mice were immunized intramuscularly with HA-M1, NA-M1 or M1 VLPs and protective immunity was evaluated by assessing lung virus loads against low (5LD50) or high (100LD50) lethal dose of homologous virus challenges. High levels of virus-specific serum IgG antibody responses were induced in mice after HA-M1 VLPs immunization, whereas low or no IgG antibody responses were detected from immunization with NA-M1 VLPs or M1 VLPs, independently. Mice that were immunized with HA-M1 VLPs showed below the limit of detection on lung virus loads against low dose (5LD50) of challenge and significant reduction against high dose (100LD50) of challenge infection. Mice that were immunized with NA-M1 or M1 VLPs also displayed reduced lung viral loads compared to naïve control. In vitro cultures of cells from mouse spleen and bone marrow revealed that HA-M1 VLPs and NA-M1 VLPs induced higher levels of antibody-secreting cell (ASC) responses compared to naïve control, whereas M1 VLPs showed no ASC responses. HA-M1, NA-M1 or M1 VLPs immunization demonstrated varying degree of protection with respect to body weight changes and survival rates, which are consistent with the levels of antibody responses in sera and ASC responses from spleen and bone marrow.

The resistance against Trichinella spiralis infection induced by primary infection with respiratory syncytial virus.

Human infections with Trichinella spiralis and respiratory syncytial virus (RSV) are common, as T. spiralis infections are re-emerging in various parts of the world and RSV infections remain a threat for infants. Yet, studies investigating the relationship pertaining to the two are severely lacking. In particular, immune response induction via RSV and T. spiralis remain largely elusive. Here, we investigated the resistance against T. spiralis infection induced upon primary infection with RSV. RSV, notorious for causing severe inflammatory reaction in the lungs, were intranasally infected, followed with a T. spiralis infection in mice. Our results revealed that primary RSV infection in mice significantly raised T. spiralis-specific and total IgE, IgG and its subclass antibody responses upon T. spiralis challenge infection (RSV-Ts). Blood eosinophil levels were decreased in RSV-Ts, accompanied with significant increase in both Th1 and Th2 cytokines. Antibodies generated against RSV in RSV-infected mice were found to react with T. spiralis excretory/secretory antigen, showing several bands determined through immunoblotting. RSV-Ts also had a marked reduction of T. spiralis worm burden in diaphragm. These results indicate that immune responses induced by RSV infection contribute to resistance against subsequent T. spiralis infection.

Parasite Infiltration and Apoptosis in Spleen upon Toxoplasma gondii Infection.

Toxoplasma gondii infection induces parasite infiltration and apoptosis in the spleen. However, dose-dependent parasite infiltration, apoptosis, body weight alternations and survival in mice remain largely unknown. In this study, mice were intraperitoneally infected with 10, 30 or 100 tachyzoites of T. gondii, respectively. Parasite infiltration and apoptosis in the spleen were analyzed on days 3, 7, and 9 post-infection by immunohistochemistry and flow cytometry. Significantly higher levels of T. gondii infiltration and apoptosis in the spleen were found in 30 and 100 tachyzoites infected mice compared to 10 tachyzoites infected mice on days 7 and 9 post-infection. Although 30 and 100 tachyzoites infected mice showed significant body weight loss compared to 10 tachyzoites infected mice, all of the 100, 30, and 10 tachyzoites infected mice died by days 12, 15, and 17, each respectively. Interestingly, T. gondii infiltration in 10 tachyzoites infected mice were limited to capsule area of the spleen on day 9 post-infection. Several areas of parasite infiltrations were found in the 30 tachyzoites infected mice, where noticeable levels of splenic capsule de-adhesion occurred. These results indicated that parasite infiltration and apoptosis in the spleen, as well as body weight loss (survival) are closely correlated with infection dosage. The level of T. gondii infiltration and apoptosis in the spleen and splenic de-adhesion were dependent on the parasite dose.

Previous Infection with Plasmodium berghei Confers Resistance to Toxoplasma gondii Infection in Mice.

Both Plasmodium spp. and Toxoplasma gondii are important apicomplexan parasites, which infect humans worldwide. Genetic analyses have revealed that 33% of amino acid sequences of inner membrane complex from the malaria parasite Plasmodium berghei is similar to that of Toxoplasma gondii. Inner membrane complex is known to be involved in cell invasion and replication. In this study, we investigated the resistance against T. gondii (ME49) infection induced by previously infected P. berghei (ANKA) in mice. Levels of T. gondii-specific IgG, IgG1, IgG2a, and IgG2b antibody responses, CD4+ and CD8+ T cell populations were found higher in the mice infected with P. berghei (ANKA) and challenged with T. gondii (ME49) compared to that in control mice infected with T. gondii alone (ME49). P. berghei (ANKA) + T. gondii (ME49) group showed significantly reduced the number and size of T. gondii (ME49) cysts in the brains of mice, resulting in lower body weight loss compared to ME49 control group. These results indicate that previous exposure to P. berghei (ANKA) induce resistance to subsequent T. gondii (ME49) infection.

Virus-like Particle Vaccine Containing Toxoplasma gondii Rhoptry Protein 13 Induces Protection against T. gondii ME49 Infection in Mice.

Toxoplasma gondii can infect humans worldwide, causing serious diseases in pregnant women and immunocompromised individuals. T. gondii rhoptry protein 13 (ROP13) is known as one of the key proteins involved in host cell invasion. In this study, we generated virus-like particles (VLPs) vaccine expressing T. gondii rhoptry ROP13 and investigated VLPs vaccine efficacy in mice. Mice immunized with ROP13 VLPs vaccine elicited significantly higher levels of T. gondii-specific IgG, IgG1, IgG2a, and IgA antibody responses following boost immunization and challenge infection, whereas antibody inductions were insignificant upon prime immunization. Differing immunization routes resulted in differing antibody induction, as intranasal immunization (IN) induced greater antibody responses than intramuscular immunization (IM) after boost and challenge infection. IN immunization induced significantly higher levels of IgG and IgA antibody responses from feces, antibody-secreting cells (ASCs), CD4+ T, CD8+ T cells and germinal center B cell responses in the spleen compared to IM immunization. Compared to IM immunization, IN immunization resulted in significantly reduced cyst counts in the brain as well as lesser body weight loss, which contributed to better protection. All of the mice immunized through either route survived, whereas all naïve control mice perished. These results indicate that the ROP13 VLPs vaccine could be a potential vaccine candidate against T. gondii infection.

Within-population genetic diversity and population structure of Plasmodium knowlesi merozoite surface protein 1 gene from geographically distinct regions of Malaysia and Thailand.

BACKGROUND: The C-terminal 42 kDa domain of Plasmodium knowlesi merozoite surface protein 1 (PkMSP1) is a potential asexual blood-stage vaccine candidate, however, only a limited number of clinical isolates have been analysed from Malaysia and no inter-country comparative diversity study has been conducted. In the present study, nucleotide diversity, haplotypes and natural selection levels of pkmsp1 in clinical samples from geographically distinct regions of Malaysia and Thailand were investigated. The overall population structure of the parasite from the region was determined. METHODS: Eleven full-length pkmsp1 sequences obtained from clinical isolates of Malaysia along with the H-strain were downloaded from the database for domain wise characterization of pkmsp1 gene. Additionally, 76 pkmsp-142 sequences from Thailand and Malaysia were downloaded from the database for intra and inter-population analysis. DnaSP 5.10 and MEGA 5.0 software were used to determine genetic diversity, polymorphism, haplotypes and natural selection. Genealogical relationships were determined using haplotype network tree in NETWORK software v5.0. Population genetic differentiation index (FST) of parasites were analysed using Arlequin v3.5. RESULTS: Sequence analysis of 11 full-length pkmsp1 sequences along with the H-strain identified 477 (8.4%) polymorphic sites, of which 107 were singleton sites. The overall diversity observed in the full-length genes were high in comparison to its ortholog pvmsp1 and the 4 variable domains showed extensive size variations. The nucleotide diversity was low towards the pkmsp1-42 compared to the conserved domains. The 19 kDa domain was less diverse and completely conserved among isolates from Malaysian Borneo. The nucleotide diversity of isolates from Peninsular Malaysia and Thailand were higher than Malaysian Borneo. Network analysis of pkmsp1-42 haplotypes showed geographical clustering of the isolates from Malaysian Borneo and grouping of isolates from Peninsular Malaysia and Thailand. Population differentiation analysis indicated high FST values between parasite populations originating from Malaysian Borneo, Peninsular Malaysia and Thailand attributing to geographical distance. Moderate genetic differentiation was observed for parasite populations from Thailand and Peninsular Malaysia. Evidence of population expansion and purifying selection were observed in all conserved domains with strongest selection within the pkmsp1-42 domain. CONCLUSIONS: This study is the first to report on inter country genetic diversity and population structure of P. knowlesi based on msp1. Strong evidence of negative selection was observed in the 42 kDa domain, indicating functional constrains. Geographical clustering of P. knowlesi and moderate to high genetic differentiation values between populations identified in this study highlights the importance of further evaluation using larger number of clinical samples from Southeast Asian countries.

Virus-Like Particles Expressing Toxoplasma gondii Rhoptry Protein 18 Induces Better Protection Than Rhoptry Protein 4 against T. gondii Infection.

Toxoplasma gondii is a ubiquitous protozoan parasite responsible for causing toxoplasmosis. Preventive measures for toxoplasmosis are currently lacking and as such, development of novel vaccines are of urgent need. In this study, we generated 2 virus-like particles (VLPs) vaccines expressing T. gondii rhoptry protein 4 (ROP4) or rhoptry protein 18 (ROP18) using influenza matrix protein (M1) as a core protein. Mice were intranasally immunized with VLPs vaccines and after the last immunization, mice were challenged with ME49 cysts. Protective efficacy was assessed and compared by determining serum antibody responses, body weight changes and the reduction of cyst counts in the brain. ROP18 VLPs-immunized mice induced greater levels of IgG and IgA antibody responses than those immunized with ROP4 VLPs. ROP18 VLPs immunization significantly reduced body weight loss and the number of brain cysts in mice compared to ROP4 VLPs post-challenge. These results indicate that T. gondii ROP18 VLPs elicited better protective efficacy than ROP4 VLPs, providing important insight into vaccine design strategy.