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Flavivirus reporters provide a robust tool for viral pathogenesis studies, anti-viral drug screening, disease diagnosis and functional antibody assays. In this study, we generated a luciferase-secreting, single-round reporter virus by replacing the capsid coding region in a DENV-2 genome with the secretory form of Lucia luciferase gene to produce infectious viral particles in a stable capsid-expressing mosquito cell line. Replication of the reporter virus in trans-complementing mosquito cells was sustained for up to two weeks. There were strong correlations between the extracellular luciferase activity and infectious reporter virus inocula upon infection of mosquito and mammalian cell lines with graded quantities of the reporter virus. A set of anti-E and anti-prM monoclonal antibodies affected the infectivity of reporter virus with similar dose-effect relationships as the parent virus. This simplified version of DENV-2 reporter provides a rapid and reliable method for the detection of neutralizing and infection-enhancing antibodies against dengue virus.

The objectives of this study were to develop a method for concentrating rotavirus, to assess the detection rate, and to characterize the genotype of naturally occurring rotavirus in bivalve shellfish species; including oysters (Saccostrea forskali), cockles (Anadara nodifera), and mussels (Perna viridis). The results demonstrated that an adsorption-twice elution-extraction method was less-time consuming method of concentrating the spiked rotavirus, yielding high sensitivity of 1.14 genome copies/g of digestive tissues from all three shellfish species, as detected using an RT-nested PCR. In seeding experiments, rotavirus as low as 1.39 genome copies was able to be detected in 4 g of digestive tissues or per sample. In the period of August 2011 to July 2012, of the 300 bivalve shellfish samples collected and tested, 24 (8.0%) were found to be contaminated with rotavirus, the figures being: oysters, 13/100 samples; mussels, 10/100 samples; and cockles, 1/100 samples. By DNA sequencing of the RT-nested PCR products and phylogenetic analysis, the rotaviruses detected were classified into G1, lineage II (4 samples); G3 (10 samples): lineage I (3 samples), lineage IIIc (3 samples), lineage IIId (3 samples), lineage IV (1 sample); G9 (6 samples); and G12, lineage III (1 sample). These findings suggest that this virus concentration method provides high sensitivity for the detection of rotavirus from the three bivalve shellfish species. The prevalence of rotavirus and the identified genotypes contribute to the molecular epidemiology of rotavirus in different shellfish species.

This study aimed to investigate the antibody responses in mice immunized with Gnathostoma spinigerum crude antigen (GsAg) incorporated with the combined adjuvant, a synthetic oligonucleotide containing unmethylated CpG motif (CpG ODN 1826) and a stable water in oil emulsion (Montanide ISA720). Mice immunized with GsAg and combined adjuvant produced all antibody classes and subclasses to GsAg except IgA. IgG2a/2b/3 but not IgG1 subclasses were enhanced by immunization with CpG ODN 1826 when compared with the control groups immunized with non-CpG ODN and Montanide ISA or only with Montanide ISA, suggesting a biased induction of a Th1-type response by CpG ODN. After challenge infection with live G. spinigerum larvae, the levels of IgG2a/2b/3 antibody subclasses decreased immediately and continuously, while the IgG1 subclass remained at high levels. This also corresponded to a continuous decrease of the IgG2a/IgG1 ratio after infection. Only IgM and IgG1 antibodies, but not IgG2a/2b/3, were significantly produced in adjuvant control groups after infection. These findings suggest that G. spinigerum infection potently induces a Th2-type biased response.

Development of a vaccine that targets blood-stage malaria parasites is imperative if we are to sustainably reduce the morbidity and mortality caused by this infection. Such a vaccine should elicit long-lasting immune responses against conserved determinants in the parasite population. Most blood-stage vaccines, however, induce protective antibodies against surface antigens, which tend to be polymorphic. Cell-mediated responses, on the other hand, offer the theoretical advantage of targeting internal antigens that are more likely to be conserved. Nonetheless, few of the current blood-stage vaccine candidates are able to harness vigorous T cell immunity. Here, we present what we believe to be a novel blood-stage whole-organism vaccine that, by combining low doses of killed parasite with CpG-oligodeoxynucleotide (CpG-ODN) adjuvant, was able to elicit strong and cross-reactive T cell responses in mice. Our data demonstrate that immunization of mice with 1,000 killed parasites in CpG-ODN engendered durable and cross-strain protection by inducing a vigorous response that was dependent on CD4+ T cells, IFN-gamma, and nitric oxide. If applicable to humans, this approach should facilitate the generation of robust, cross-reactive T cell responses against malaria as well as antigen availability for vaccine manufacture.

It is well known that exposure to one antigen can modulate the immune responses that develop following exposure to closely related antigens. It is also known that the composition of the repertoire can be skewed to favor epitopes shared between a current infection and a preceding one, a phenomenon referred to as “original antigenic sin.” It was of interest, therefore, to investigate the antibody response that develops following exposure to the malaria vaccine candidate homologuePlasmodium yoeliiMSP119in mice that had previously experienced malaria infection and vice versa. In this study, preexposure of mice toPlasmodium yoeliielicited native anti-MSP119antibody responses, which could be boosted by vaccination with recombinant MSP119. Likewise, infection of MSP119-primed mice withP. yoeliiled to an increase of anti-MSP119antibodies. However, this increase was at the expense of antibodies to parasite determinants other than MSP119. This change in the balance of antibody specificities significantly affected the ability of mice to withstand a subsequent infection. These data have particular relevance to the possible outcome of malaria vaccination for those situations where the vaccine response is suboptimal and suggest that suboptimal vaccination may in fact render the ultimate acquisition of natural immunity more difficult.

Dendritic cells (DCs) initiate innate and adaptive immune responses including those against malaria. Although several studies have shown that DC function is normal during malaria, other studies have shown compromised function. To establish why these studies had different findings, we examined DCs from mice infected with two lethal species of parasite, Plasmodium berghei or P. vinckei, and compared them to DCs from nonlethal P. yoelii 17XNL or P. chabaudi infections. These studies found that DCs from only the lethal infections became uniformly mature 7 days after infection and were functionally impaired as they were unable to endocytose latex particles, secrete IL-12, or present OVA to transgenic OTII T cells. These changes coincided with a peak in levels of systemic TNF-α. Because TNF-α is known to mature DCs, we used TNF-KO mice to determine the role of this cytokine in the loss of DC function. In the TNF-KO mice, phenotype, Ag presentation, and IL-12 secretion by DCs were restored to normal following both lethal infections. This study shows that the systemic production of TNF-α contributes to poor DC function during lethal infections. These studies may explain, at least in part, immunosuppression that is associated with malaria.

Summary Background The ability of T cells, acting independently of antibodies, to control malaria parasite growth in people has not been defined. If such cell-mediated immunity was shown to be effective, an additional vaccine strategy could be pursued. Our aim was to ascertain whether or not development of cell-mediated immunity to Plasmodium falciparum blood-stage infection could be induced in human beings by exposure to malaria parasites in very low density. Methods We enrolled five volunteers from the staff at our research institute who had never had malaria. We used a cryopreserved inoculum of red cells infected with P falciparum strain 3D7 to give them repeated subclinical infections of malaria that we then cured early with drugs, to induce cell-mediated immune responses. We tested for development of immunity by measurement of parasite concentrations in the blood of volunteers by PCR of the multicopy gene STEVOR and by following up the volunteers clinically, and by measuring antibody and cellular immune responses to the parasite. Findings After challenge and a extended period without drug cure, volunteers were protected against malaria as indicated by absence of parasites or parasite DNA in the blood, and absence of clinical symptoms. Immunity was characterised by absence of detectable antibodies that bind the parasite or infected red cells, but by the presence of a proliferative T-cell response, involving CD4+ and CD8+ T cells, a cytokine response, consisting of interferon γ but not interleukin 4 or interleukin 10, induction of high concentrations of nitric oxide synthase activity in peripheral blood mononuclear cells, and a drop in the number of peripheral natural killer T cells. Interpretation People can be protected against the erythrocytic stage of malaria by a strong cell-mediated immune response, in the absence of detectable parasitespecific antibodies, suggesting an additional strategy for development of a malaria vaccine

The immune response to malaria parasites includes T cell responses that reduce parasites by effector T cell responses and by providing help for antibody responses. Some parasites are more sensitive to antibody and others are more sensitive to cell-mediated immunity. We demonstrate that cultured CD4+T cells that produce interferon γ and interleukin 2, but not interleukin 4, in response to stimulation with the rodent parasitePlasmodium bergheican reduce but not eliminate parasitesin vivoafter adoptive transfer. Although cells can persistin vivofor up to 9 months in uninfected mice, infection results in elimination of up to 99% of specific T cells in different tissues, as judged by tracking T cells labeled with the fluorescent dye 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester. T cells specific for ovalbumin are unaffected.In vivoactivation and division of transferred T cellsper seare not responsible for deletion because T cells positive for 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester divide up to six times within 7 days in uninfected mice and are not deleted. Understanding the factors responsible for parasite-mediated specific deletion of T cells would enhance our knowledge of parasite immunity.

To investigate the efficacy of a vaccine formulation of the 19 kDa conserved carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (PyMSP1(19)) formulated with CpG ODN 1826 and Montanide ISA51 or ISA720 when used to immunize mice by a single injection.Groups of BALB/c mice were immunized parenterally with one, two or four injections with PBS or PyMSP1(19) formulated with CpG ODN in ISA51 or ISA720. Sera were collected weekly and assessed for total IgG and IgG subclass titers. Protection was tested by challenge infection with P. yoelii YM.Interestingly, single injection immunization showed the same kinetics of antibody responses as two- or four-injection immunization. However, the peak antibody response induced by PyMSP1(19) in CpG ODN and ISA51 appeared earlier than that induced by PyMSP1(19) in CpG ODN and ISA720 (28 days vs 41 days). At day 63 after the first injection, the PyMSP1(19)-specific IgG antibody levels by single injection and four-injection immunizations were not different. However, the levels of the IgG2a antibody subclass were significantly lower by single injection immunization with PyMSP1(19) in CpG ODN and ISA720. The antibodies were sustained at high levels for at least 20 weeks. After challenge infection, all mice immunized by a single injection of PyMSP1(19) in CpG ODN and ISA51 survived with low-grade parasitemia, while 50% of mice immunized with PyMSP1(19) in CpG ODN and ISA720 died with high levels of parasitemia.These findings suggest that MSP1(19) immunization by a single injection can induce protective immunity, particularly when formulated with an appropriate strong adjuvant.

Naturally acquired immune response to C-terminal region of Plasmodium vivax merozoite surface protein1 (PvMSP1) in 200 individuals with recent clinical episodes of malaria from malaria endemic areas along Thai-Myanmar border in the west and Thai-Cambodia border in the east of Thailand was evaluated by enzyme-linked immunosorbent assay (ELISA). The anti-PvMSP1-IgG antibody was observed in 110 individuals (55%). Among IgG responders, IgG1 coexpressed with IgG3 were the predominant subclasses. The levels of anti-PvMSP1 total IgG, IgG1 and IgG3 antibody response seem to be increased with age although no detectable significant correlation was found (r = 0.004, p = 0.484 for total IgG; r = 0.035, p = 0.386 for IgG1; r = -0.600, p = 0.142 for IgG2; r = 0.077, p = 0.227 for IgG3; r = 0.664, p = 0.051 for IgG4). However, the mean level of specific total IgG was highest in the age group of40 years. These levels of either specific total IgG or each IgG isotype did not vary among individuals with different malaria episodes. A higher level of specific total IgG, IgG1 and IgG3 antibody response related with the lower of parasitemia density was observed although no significant correlation was found. Our data indicate that individuals exposed to vivax malaria in Thailand developed antibodies to the potential candidate vaccine antigen, PvMSP1 (C-terminal).

Interaction between different bacterial plaque pathogens and dendritic cells may induce different types of T helper (Th) cell response, which is critical in the pathogenesis of periodontitis. In this study we investigated the effects of lipopolysaccharide (LPS) from Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans on human monocyte-derived dendritic cells (Mo-DCs) with respect to co-stimulatory molecule expression, cytokine production and Th cell differentiation. Unlike Escherichia coli and A. actinomycetemcomitans LPS, P. gingivalis LPS induced only low levels of CD40, CD80, HLA-DR and CD83 expression on Mo-DCs. LPS from both bacteria induced considerably lower TNF-alpha and IL-10 than did E. coli LPS. LPS from all three bacteria induced only negligible IL-12 production. In a human mixed-leukocyte reaction, and in an ovalbumin-specific T cell response assay in mice, both types of LPS suppressed IFN-gamma production. In conclusion, stimulation by P. gingivalis LPS and A. actinomycetemcomitans LPS appears to bias Mo-DCs towards Th2 production.

Merozoite surface protein 1 (MSP1) is the major protein on the surface of the plasmodial merozoite, and its carboxy terminus, the 19-kDa fragment (MSP1 19 ), is highly conserved and effective in induction of a protective immune response against malaria parasite infection in mice and monkeys. However, the duration of the immune response has not been elucidated. As such, we immunized BALB/c mice with a standard four-dose injection of recombinant Plasmodium yoelii MSP1 19 formulated with Montanide ISA51 and CpG oligodeoxynucleotide (ODN) and monitored the MSP1 19 -specific antibody levels for up to 12 months. The antibody titers persisted constantly over the period of time without significant waning, in contrast to the antibody levels induced by immunization with Freund's adjuvant, where the antibody levels gradually declined to significantly lower levels 12 months after immunization. Investigation of immunoglobulin G (IgG) subclass longevity revealed that only the IgG1 antibody level (Th2 type-driven response) decreased significantly by 6 months, while the IgG2a antibody level (Th1 type-driven response) did not change over the 12 months after immunization, but the boosting effect was seen in the IgG1 antibody responses but not in the IgG2a antibody responses. After challenge infection, all immunized mice survived with negligibly patent parasitemia. These findings suggest that protective immune responses to MSP1 19 following immunization using oil-based Montanide ISA51 and CpG ODN as an adjuvant are very long-lasting and encourage clinical trials for malaria vaccine development.

Protective cellular immune responses depend on MHC presentation of pathogen-derived Ag fragments. MHC diversity renders this process sensitive to point mutations coding for altered amino acid sequence of the short target Ag-derived peptides epitopes. Thus, in a given host, a pathogen with an altered epitope sequence will be more likely to escape detection and elimination by the immune system. At a population level, selection by immune pressure will increase the likelihood of polymorphism in important pathogen antigenic epitopes. This mechanism of immune evasion is found in viruses and other pathogens. The detection of polymorphic hot spots in an Ag is often taken as a strong indication of its role in protective immunity. We provide evidence that polymorphisms in the T cell epitopes of a malaria vaccine candidate are unlikely to have been selected by immune pressure in the human host.

Allelic variation in the Plasmodium falciparum circumsporozoite protein (CS) gene has been determined by sequencing the immunodominant T-cell epitopes, Th2R and Th3R, from 95 isolates from two malaria-endemic areas in the west of Thailand. Comparison with a reference sequence revealed only non-synonymous point mutations in the two epitope regions. Point mutations were found outside these epitopes in a minority of samples, and all but four were also non-synonymous. A relatively high number of variants, 11 Th2R and 9 Th3R, were detected and comprised some that had not been previously observed. However, the Th2R*05 and the Th3R*01 allelic variants predominated, as they were found in more than 70% of the 101 sequences obtained.

The 19kDa carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (MSP1(19)), an analog of the leading falciparum malaria vaccine candidate, induces protective immunity to challenge infection when formulated with complete/incomplete Freund's adjuvant (CFA/IFA), an adjuvant unsuitable for use in humans. In this study, we investigate Montanide ISA51 and Montanide ISA720 as well as CpG oligodeoxynucleotide (ODN) as adjuvants for induction of immunity to MSP1(19). Mice immunized with MSP1(19) adjuvanted with Montanide ISA51 were protected even though some mice experienced low-grade parasitemia before resolving the infection. Mice immunized with MSP1(19) adjuvanted with Montanide ISA720 showed delayed patent parasitemia with all mice ultimately succumbing to infection. Interestingly, when the synthetic CpG ODN 1826 was included in either Montanide formulation, mice were completely protected with no parasites detected in the blood. MSP1(19)-specific antibodies in MSP1(19)-immunized mice adjuvanted with Montanide ISA51 or Montanide ISA720 showed predominantly IgG1 antibody and low levels of IgG2a. CpG ODN 1826 significantly enhanced both IgG1 and IgG2a antibody responses in Montanide ISA51-adjuvanted mice but significantly enhanced only the IgG2a antibody response in Montanide ISA720-adjuvanted mice. To investigate the relative roles of antibody and CD4(+) T cells in protection, MSP1(19)-immunized mice adjuvanted with Montanide ISA720 and CpG ODN 1826 were depleted of CD4(+) T cells just prior to challenge. Results showed that three of nine immunized/T cell depleted mice died following infection. These results suggest that antibody and CD4(+) T cells are critical for protection following immunization with MSP1(19) adjuvanted with Montanide and CpG ODN and that the formulation of a human malaria vaccine candidate in Montanide ISA720 or ISA51 together with human compatible CpG ODN would be useful for improving efficacy.

Although there is good evidence that immunity to the blood stages of malaria parasites can be mediated by different effector components of the adaptive immune system, target antigens for a principal component, effector CD4+T cells, have never been defined. We generated CD4+T cell lines to fractions of native antigens from the blood stages of the rodent parasite,Plasmodium yoelii, and identified fraction-specific T cells that had a Th1 phenotype (producing IL-2, IFN-γ, and tumor necrosis factor-α, but not IL-4, after antigenic stimulation). These T cells could inhibit parasite growth in recipient severe combined immunodeficient mice. N-terminal sequencing of the fraction showed identity with hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT). Recombinant HGXPRT from the human malaria parasite,Plasmodium falciparum, activated the T cellsin vitro, and immunization of normal mice with recombinant HGXPRT reduced parasite growth rates in all mice after challenge.

CD4+ T cells have been implicated in immunity to the blood stages of malaria and cytokines associated with both monocyte and T cell activation have been implicated in disease. To determine whether specific T cells capable of inhibiting parasite growth can also mediate pathology we have transfused populations of Plasmodium berghei-specific T cells into normal and immunodeficient naive mice. We observed that they could inhibit parasite growth but were unable to save the animals which exhibited significantly greater anaemia and weight loss than control infected animals receiving either no T cells or T cells specific for ovalbumin. T cell-dependent tomour necrosis factor (TNF)alpha was a critical component in both parasite killing and disease promotion. Experiments with blocking antibodies demonstrated that all T-cell mediated antiparasitic immunity and all T-cell mediated weight loss was TNF-dependent. Blocking TNF-alpha in mice that received parasite-specific T cells prolonged the survival of the mice. Nitric oxide demonstrated no antiparasite effect, but was involved in the regulation of T-cell mediated weight loss. The data thus show that while parasite-specific CD4+ T cells can significantly limit parasite growth, such an effect need not be beneficial to the host, and that TNF-alpha and nitric oxide are critical effector molecules operating downstream of parasite-specific T cells in both immunity and disease.

Variable protection against malaria blood-stage infection has been demonstrated in mice following parenteral immunization with the highly conserved 19 kD carboxylterminal fragment of the merozoite surface protein-1 (MSP119) using CFA/IFA and other adjuvants. Here we show that intranasal immunization of BALB/C mice with yeast expressed Plasmodium yoelii MSP119 plus a mixture of native and recombinant cholera toxin B subunit, could induce serum MSP119-specific antibodies at titres ranging from 20 000 to 2 560 000. The Ig subclass responses were predominantly G1 and G2b. Intranasal immunization led to protection following challenge (peak parasitaemia1%) in mice with the highest MSP119-specific titre (/= 640 000). In two of the three protected mice, a peak parasitaemia of 0.1%-1% was followed by a boost of the antibody response whereas one of the three protected mice did not boost its antibody response after a peak parasitaemia of 0.02%. In unprotected mice, antibody levels rose, then fell, following the detection of parasites in the peripheral blood. CD4+ T cell-depletion abrogated the ability of the mice to boost their antibody response following challenge. These data demonstrate the potential for intranasal immunization with MSP119 to protect against malaria.

The development of IgG subclass-specific antibody responses to Plasmodium berghei in spleen-chimeric rats were monitored to determine if there was any relationship between IgG subset profiles and resistance. Strongly immune eusplenic rats respond to challenge with P. berghei by producing high levels of parasite-specific IgG2a, IgG2b and IgG2c but only modest levels of IgG1. Splenectomy profoundly affects the antibody response to infection. Thus, in splenectomized immunized rats, which harbour a chronic parasitaemia of 1%, the IgG2a, IgG2b and IgG2c responses peak 1 week later than in eusplenic immunized rats although the size of the peak is similar. More marked effects are apparent in the IgG1 response, the magnitude of which is far greater in splenectomized immunized rats than eusplenic immunized rats. Similar antibody profiles are seen in splenectomized immunized rats transplanted with a naive spleen. In contrast, splenectomized naive rats receiving either a transplant of a spleen from an immune rat or a transfer of immune spleen cells have high levels of IgG2a, IgG2b and IgG2c but modest levels of IgG1. However, only the former group of rats completely clears the parasite, the latter maintaining a chronic 1% parasitaemia. Thus, although complete resistance to P. berghei is always associated with high levels of parasite-specific IgG2a, IgG2b and IgG2c plus modest levels of IgG1, this is not a sufficient set of conditions to guarantee complete immunity. The IgG subset profile may be related to cytokine production; IFN-gamma was detected in the sera of rats receiving spleens from rats immune to P. berghei (modest IgG1 responses) but not in rats receiving spleens from naive animals (pronounced IgG1 responses).

A number of reports have suggested that the spleen plays a key role in the regulation of immunity to malaria but the role, if any, of other tissues is less clear. Furthermore, numerous functional changes occur in the spleen following malaria infection and it is not known whether the spleen's role relates primarily to its content of malaria-specific lymphocytes or to the altered structure and function that has occurred. To address these issues we have generated splenic chimeras by transplanting spleens between Plasmodium berghei-immune and naive rats. In the absence of a functional spleen, specific immune responses from both isolated splenic and non-splenic cells can partially control infection. However, an immune spleen in a naive rat can solidly protect the animal from malaria and a normal spleen in an otherwise immune rat can provide enhanced protection over the non-splenic state. Thus, in the presence of functional splenic architecture both splenic and non-splenic malaria-specific lymphocytes operate more effectively. However, these studies do demonstrate an important role for non-splenic tissue in immunity at least for P. berghei in the rat. The study could have important implications for induction of protective immune responses by vaccination and suggests that malaria-specific lymphocyte responses induced in the periphery following vaccination could interact with parasites in both spleen-dependent and spleen-independent ways.