Helminth infection reactivates latent γ-herpesvirus via cytokine competition at a viral promoter.

Mammals are coinfected by multiple pathogens that interact through unknown mechanisms. We found that helminth infection, characterized by the induction of the cytokine interleukin-4 (IL-4) and the activation of the transcription factor Stat6, reactivated murine γ-herpesvirus infection in vivo. IL-4 promoted viral replication and blocked the antiviral effects of interferon-γ (IFNγ) by inducing Stat6 binding to the promoter for an important viral transcriptional transactivator. IL-4 also reactivated human Kaposi's sarcoma-associated herpesvirus from latency in cultured cells. Exogenous IL-4 plus blockade of IFNγ reactivated latent murine γ-herpesvirus infection in vivo, suggesting a "two-signal" model for viral reactivation. Thus, chronic herpesvirus infection, a component of the mammalian virome, is regulated by the counterpoised actions of multiple cytokines on viral promoters that have evolved to sense host immune status.

IL-4(-/-) mice with lethal Mesocestoides corti infections--reduced Th2 cytokines and alternatively activated macrophages.

Protection against Mesocestoides corti, a cestode that invades vital organs, is dependent on the production of IL-4, as IL-4(-/-) mice were found to have higher parasite burdens when compared with wild-type mice. The goal of this study was to investigate the role of IL-4 in immunity to M. corti, focusing on the immunological profile and on potential mediators of pathology. IL-4(-/-) mice infected with M. corti showed 100% mortality by 32 days, whereas wild-type mice survived for approximately 1 year. Parasite burdens were significantly increased in the liver, peritoneal, and thoracic cavities of IL-4(-/-) mice, associated with impaired recruitment of inflammatory cells and a reduction in monocytes and macrophages. IL-5 production by splenocytes and expression in liver tissue was decreased in infected IL-4(-/-) mice compared with wild-type mice. In contrast, IL-4(-/-) mice produced increased amounts of IFNgamma and TNFalpha. Alternatively activated macrophages were a major feature of liver granulomas in wild-type mice evidenced by Arginase I expression, while livers from infected IL-4(-/-) mice showed impaired alternative macrophage activation without increased classical macrophage activation. Thus, lethality during M. corti infection of IL-4(-/-) mice is associated with decreased Th2 cytokines, increased Th1 cytokines and impairment of alternatively activated macrophages.

Reverse genetics and the study of the immune response to schistosomes.

The sequencing of the schistosome genome and the establishment of techniques for RNAi and transient transfection in these parasites have opened the door for a reverse genetics approach to studying schistosomes. One of the most intriguing aspects of schistosome biology is the interaction of these parasites with the immune system. The immune response underlies the ability of the host to survive while infected and to eventually develop resistance to further infection. However, it is also instrumental in the development of disease due to its role orchestrating granuloma formation around tissue-trapped parasite eggs. While schistosomes have clearly evolved mechanisms for evading host immune responses, their normal development is, paradoxically, also dependent upon the presence of a normal immune system. This article will review recent advances in the development of tools for studying gene function in schistosomes, and discuss how these new tools may be exploited to investigate issues of key importance in the interaction of schistosomes with the host immune system.

Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of L-arginine metabolism.

Type 2 cytokines regulate fibrotic liver pathology in mice infected with Schistosoma mansoni. Switching the immune response to a type 1-dominant reaction has proven highly effective at reducing the pathologic response. Activation of NOS-2 is critical, because type 1-deviated/NO synthase 2 (NOS-2)-deficient mice completely fail to control their response. Here, we demonstrate the differential regulation of NOS-2 and arginase type 1 (Arg-1) by type 1/type 2 cytokines in vivo and for the first time show a critical role for arginase in the pathogenesis of schistosomiasis. Using cytokine-deficient mice and two granuloma models, we show that induction of Arg-1 is type 2 cytokine dependent. Schistosome eggs induce Arg-1, while Mycobacterium avium-infected mice develop a dominant NOS-2 response. IFN-gamma suppresses Arg-1 activity, because type 1 polarized IL-4/IL-10-deficient, IL-4/IL-13-deficient, and egg/IL-12-sensitized animals fail to up-regulate Arg-1 following egg exposure. Notably, granuloma size decreases in these type-1-deviated/Arg-1-unresponsive mice, suggesting an important regulatory role for Arg-1 in schistosome egg-induced pathology. To test this hypothesis, we administered difluoromethylornithine to block ornithine-aminodecarboxylase, which uses the product of arginine metabolism, L-ornithine, to generate polyamines. Strikingly, granuloma size and hepatic fibrosis increased in the ornithine-aminodecarboxylase-inhibited mice. Furthermore, we show that type 2 cytokine-stimulated macrophages produce proline under strict arginase control. Together, these data reveal an important regulatory role for the arginase biosynthetic pathway in the regulation of inflammation and demonstrate that differential activation of Arg-1/NOS-2 is a critical determinant in the pathogenesis of granuloma formation.

Role of gamma interferon in the pathogenesis of severe schistosomiasis in interleukin-4-deficient mice.

In the absence of interleukin-4 (IL-4), infection with Schistosoma mansoni leads to a severe fatal disease rather than the chronic survivable condition that occurs in wild-type (WT) mice. Because the sustained production of NO most closely correlates to weight loss and fatality in infected IL-4(-/-) mice and because gamma interferon (IFN-gamma) is an important inducer of inducible NO synthase, infected IL-4(-/-) mice were treated with anti-IFN-gamma antibodies to determine the role of IFN-gamma during schistosomiasis in WT and IL-4(-/-) animals. When IFN-gamma was neutralized, Th2 responses were enhanced and NO production was reduced in both WT and IL-4(-/-) mice. The decreased NO production correlated with a rescue of proliferation in splenocytes from infected IL-4(-/-) mice. Furthermore, the neutralization of IFN-gamma in vivo improved the gross appearance of the liver and led to a reduction in granuloma size in infected IL-4(-/-) but not WT mice. However, the neutralization of IFN-gamma in vivo did not affect the development of severe disease in infected IL-4(-/-) mice. These results suggest that while the increased production of IFN-gamma does lead to some of the pathology observed in infected IL-4(-/-) mice, it is not ultimately responsible for cachexia and death.

CD8- dendritic cell activation status plays an integral role in influencing Th2 response development.

Whether dendritic cells (DC) play a passive or active role in Th2 response induction is poorly understood. In this study, we show that CD8- DC pulsed with Th2-polarizing Ag (soluble egg Ag (SEA)) from Schistosoma mansoni potently stimulate Th2 responses in vivo and in vitro while failing to undergo a conventional maturation process. Thus, in contrast to DC pulsed with the Th1 response inducing Ag Propionebacterium acnes, SEA-exposed DC exhibit a phenotype that is most similar to that of immature DC, failing to up-regulate expression of CD40, CD54, CD80, CD86, or OX40L; producing no detectable IL-4, IL-10, or IL-12; and displaying only a minor increase in MHC class II expression. Importantly, in vitro derived DC exposed to SEA were phenotypically similar to CD8- DC isolated from active S. mansoni infection. By discriminating between different types of pathogen and responding appropriately, CD8- DC play a major role in the decision process to mount either a Th1 or Th2 response.

Interleukin-12 promotes pathologic liver changes and death in mice coinfected with Schistosoma mansoni and Toxoplasma gondii.

We previously demonstrated that mice concurrently infected with Schistosoma mansoni and Toxoplasma gondii undergo accelerated mortality which is preceded by severe liver damage. Abnormally high levels of serum tumor necrosis factor alpha (TNF-alpha) in the dually infected mice suggested a role for this and related proinflammatory mediators in the pathologic alterations. In order to evaluate the factors involved in increased inflammatory-mediator production and mortality, interleukin-12(-/-) (IL-12(-/-)) mice were coinfected with S. mansoni and T. gondii, and survival and immune responses were monitored. These IL-12(-/-) mice displayed decreased liver damage and prolonged time to death relative to wild-type animals also coinfected with these parasites. Relative to the response of cells from the coinfected wild-type animals, levels of TNF-alpha, gamma interferon, and NO produced by splenocytes from coinfected IL-12(-/-) mice were reduced, and levels of IL-5 and IL-10 were increased, with the net result that the immune response of the dually infected IL-12(-/-) mice was similar to that of the wild-type mice infected with S. mansoni alone. While dually infected wild-type animals succumb in the absence of overt parasitemia, the delayed death in the absence of IL-12 is associated with relatively uncontrolled T. gondii replication. These data support the view that S. mansoni-infected mice are acutely sensitive to infection with T. gondii as a result of their increased hepatic sensitivity to high levels of proinflammatory cytokines; IL-12 and TNF-alpha are implicated in this process.

IL-4 plays a crucial role in regulating oxidative damage in the liver during schistosomiasis.

Liver enlargement and hepatocyte proliferation, normal responses in wild-type (WT) mice infected with the parasitic helminth Schistosoma mansoni, were found to be severely impaired in infected IL-4(-/-) mice. Compared with WT mice, increased levels of O(2)(-), NO, and the more highly reactive ONOO(-) were detected in the liver and produced by lesional cells isolated from liver granulomas of infected IL-4(-/-) mice. Concurrently, antioxidant defenses in the liver, specifically catalase levels, diminished dramatically during the course of infection in these animals. This contrasted to the situation in infected WT mice, where catalase levels remained as high as those in normal mice. Actual levels of reactive oxygen and nitrogen intermediates in the livers of infected IL-4(-/-) animals are thus likely to be considerably higher than those in the livers of infected WT mice. To determine whether these changes contributed to the development of the more severe disease that characterizes infection in the IL-4(-/-) animals, we treated infected IL-4(-/-) mice with uric acid, a potent scavenger of ONOO(-). This resulted in significantly increased hepatocyte proliferation, decreased morbidity, and prolonged survival. Taken together, these data indicate that IL-4 is playing a protective role during schistosomiasis by controlling the tight regulation of the generation of reactive oxygen and nitrogen intermediates in the liver.

Severe schistosomiasis in the absence of interleukin-4 (IL-4) is IL-12 independent.

An interleukin-4 (IL-4)-dependent Th2 response allows wild-type mice to survive infection with the parasite Schistosoma mansoni. In the absence of IL-4, infected mice mount a Th1-like proinflammatory response, develop severe disease, and succumb. Neither the Th1 response nor morbidity is IL-12 dependent in this system.

Role of IL-6 in directing the initial immune response to schistosome eggs.

The eggs of Schistosoma mansoni are strong inducers of a Th2 response, and previous work has shown that Ag-specific IL-6 is produced within 24 h after the injection of eggs into mice. Investigations to determine the role of IL-6 in orchestrating the early response to schistosome eggs have revealed that IL-12 is rapidly produced in lymph node cell cultures from egg-injected mice. This "early" IL-12 primes for the production of IL-6 and IFN-gamma, for in IL-12-/- mice egg injection fails to stimulate increased production of either of these cytokines. Furthermore, IL-6 also up-regulates IL-10 production which, together with IL-6, negatively regulates IL-12 and IFN-gamma production. Finally, IL-10 down-regulates the production of its inducer, IL-6. These data indicate that the anti-inflammatory role of IL-6 may be effected through negative regulation of type 1 (IFN-gamma) and type 1-associated (IL-12) cytokines either directly (by IL-6) or indirectly (through the induction of IL-10) and suggest that one mechanism by which eggs may support the development of Th2 responses is through the negative regulation of the type 1 response.

Nitric oxide and the Th2 response combine to prevent severe hepatic damage during Schistosoma mansoni infection.

During infection with Schistosoma mansoni, NO production increases following the deposition of parasite eggs in the liver. In wild-type C57BL/6 mice, NO levels peak during the sixth week of infection and are subsequently down-regulated. Inducible NO synthase (iNOS) mRNA was found in diseased liver tissue along with TNF-alpha and IFN-gamma, which are known promoters of iNOS expression. Mice treated with aminoguanidine, a selective inhibitor of iNOS, exhibited cachexia and exacerbated liver pathology, suggesting that NO limits hepatocyte damage when the liver is first exposed to eggs. Hepatic iNOS is up-regulated in SCID mice, indicating that NO production is part of an innate response. Studies with infected highly susceptible IL-4-/- mice revealed that prolonged NO production is in itself deleterious and that a major function of the Th2 response, which is severely compromised in the absence of IL-4, is to regulate NO production. In these animals, plasma NO levels are high compared with those in infected wild-type mice and remain elevated until death. Nevertheless, the underlying importance of NO is illustrated by the finding that aminoguanidine treatment leads to more severe liver disease and reduced time to death in infected IL-4-/- mice.

Toxoplasma gondii and Schistosoma mansoni synergize to promote hepatocyte dysfunction associated with high levels of plasma TNF-alpha and early death in C57BL/6 mice.

To address the question of how the murine host responds to a prototypic type 1 cytokine inducer while concurrently undergoing a helminth-induced type 2 cytokine response, C57BL/6 strain animals with patent schistosomiasis mansoni were orally infected with the cystogenic Toxoplasma gondii strain ME49. Schistosoma mansoni infection resulted in a significantly higher mortality rate when mice were subsequently orally infected with ME49, and these animals displayed a defective IFN-gamma and NO response relative to animals infected with T. gondii alone. Plasma levels of TNF-alpha and aspartate transaminase in double-infected mice were greatly elevated relative to mice infected with either parasite alone. Consistent with the latter observation, these animals exhibited severe liver pathology, with regions of coagulative necrosis and hepatocyte vacuolization unapparent in mice carrying either infection alone. Interestingly, mean egg granuloma size was approximately 50% of that in mice with S. mansoni infection alone. The exacerbated liver pathology in coinfected mice did not appear to be a result of uncontrolled tachyzoite replication, because both parasite-specific RT-PCR analysis and immunohistochemical staining demonstrated a low number of tachyzoites in the liver. We hypothesize that mortality in these animals results from the high level of systemic TNF-alpha, which mediates a severe liver pathology culminating in death of the animal.

Interleukin 5 (IL-5) is not required for expression of a Th2 response or host resistance mechanisms during murine schistosomiasis mansoni but does play a role in development of IL-4-producing non-T, non-B cells.

During schistosomiasis, interleukin-5 (IL-5)-dependent eosinophil responses have been implicated in immunopathology, resistance to superinfection, synergistic interactions with chemotherapeutic agents, and the inductive phase of the egg-induced Th2 response. We examined these issues in IL-5-deficient (IL-5(-/-)) mice. IL-5(-/-) and wild-type (WT) mice were indistinguishable in terms of susceptibility to primary infections and the ability to resist secondary infections. Moreover, hepatic pathology was similar in both strains apart from a relative lack of eosinophils and, during chronic infection, a significantly larger mast cell component in the granulomas of IL-5(-/-) mice. Splenocyte cytokine production in response to soluble egg antigen (SEA) or anti-CD3 revealed no significant differences except for heightened tumor necrosis factor alpha production by cells from chronically infected IL-5(-/-) mice compared to WT animals. In contrast, ionomycin-stimulated non-B, non-T (NBNT) cells from IL-5(-/-) mice produced significantly smaller IL-4 amounts than did NBNT cells from WT animals. This difference was not apparent following plate-bound anti-immunoglobulin E or SEA stimulation. The absence of IL-5 failed to affect the induction of Th2 responses in naive mice. Peritoneal exudate cells recovered from egg-injected IL-5(-/-) or WT mice produced equivalent levels of IL-4 following restimulation with SEA or anti-CD3.

The absence of IL-6 does not affect Th2 cell development in vivo, but does lead to impaired proliferation, IL-2 receptor expression, and B cell responses.

The role of IL-6 in Th2 cell differentiation and response development after the injection of eggs from Schistosoma mansoni was investigated using wild-type (WT) and IL-6-/- mice. IL-6 was induced in the lymph nodes (LN) of WT mice within 24 h of egg injection, and IL-4 production by WT LN cells and CD4 T cells isolated 24 h after egg injection and stimulated in vitro was observed. In the absence of IL-6, this early production of IL-4 by LN cells and purified CD4 T cells was not abolished; although the level of IL-4 produced by IL-6-/- LN cells was similar to WT, IL-4 production by purified IL-6-/- CD4 T cells was reduced compared with WT. Despite the difference in CD4 T cell production of IL-4, the development of egg-specific Th2 cells 7 days after egg injection was not affected by the absence of IL-6. Nevertheless, Ab production was impaired and the in vitro proliferative response of whole LN cell populations, CD4 and CD8 T cells, and B cells from IL-6-/- mice was poor compared with WT. The proliferative defect in the IL-6-/- cells correlated with decreased IL-2R expression, and addition of exogenous IL-6 enhanced IL-2R expression as well as proliferation of LN cells from IL-6-/- mice. These studies demonstrate that Th2 differentiation and response development in vivo is not dependent on IL-6, but that Th-dependent and independent B cell responses are. Our results also emphasize the importance of IL-6 for lymphoproliferation, possibly through induction of IL-2R expression.

The initiation and function of Th2 responses during infection with Schistosoma mansoni.

Through experiments in which pro-inflammatory mediators have been neutralized in schistosomeinfected WT and IL-4 -/- mice we have been investigating the nature of the immune response that is required to allow survival during the period of acute disease that accompanies the onset of egg production by the parasitic worms. The developing picture is that of an early pro-inflammatory type-1 like response (characterized by the production of IFN-gamma and TNF-alpha and NO) which in the short term is able to control the deleterious effects on the liver associated with the arrival of the eggs, followed rapidly by a potent egg-induced Th2 response which simultaneously subsumes the role of protecting the liver and downregulates the production of the inflammatory mediators, thereby averting the serious consequence associated with the continued high level production of NO. Current research is directed towards: 1) understanding how pro-inflammatory mediators are protective during schistosomiasis, and 2) elucidating the underlying mechanisms through which the Th response is biased in a Th2 direction following exposure to schistosome eggs.

IL-4 protects against TNF-alpha-mediated cachexia and death during acute schistosomiasis.

To examine the role of the Th2-type response during schistosomiasis mansoni we compared disease progression in wild type (wt), and Th2-response deficient IL-4(-/-) mice. Whereas wt C57BL/6 mice tolerate infection and develop chronic disease, IL-4(-/-) C57BL/6 animals are highly susceptible, exhibiting severe acute cachexia followed by death. Data point toward morbidity in the IL-4(-/-) C57BL/6 mice being mediated by TNF-alpha, possibly through the uncontrolled production of nitric oxide in target organs such as the ileum. We propose that IL-4 prevents severe disease during schistosomiasis by regulating macrophage activation.

Schistosoma mansoni in IL-4-deficient mice.

Immunopathology and immune responses to Schistosoma mansoni were examined in IL-4 -/- mice. IL-5 and IL-10 production by lymphoid cells stimulated with soluble egg antigen (SEA), peripheral eosinophilia and serum levels of soluble IL-4 receptor but not IgE were all significantly elevated over background normal levels in IL-4 -/- mice as a result of infection. Additionally, IL-10 and IL-5 in addition to IL-2 and IFN-gamma transcripts were equally evident in diseased liver tissue from infected IL-4 -/- and wild-type mice. Nevertheless, analysis of antigen-stimulated IL-2, IL-4, IL-5, IL-10 and IFN-gamma production by lymphoid organ cells from infected or egg-injected IL-4 -/- mice revealed a more Th1-like pattern of cytokine production (IFN-gamma > IL-5) than in (wild-type) mice in which a stronger type 2 response to SEA was detectable (IL-4, IL-5 > IFN-gamma). Despite this, at 8 and 16 weeks after infection, liver pathology, as indicated by the size, cellularity, cellular composition and collagen content of granulomas, was similar in IL-4 -/- and wild-type animals. As in wild-type animals, granuloma size at week 16 was smaller than at week 8, indicating that modulation had occurred in the absence of IL-4. Differences in pathology were seen only when eggs were experimentally embolized to the lungs, in which case IL-4 -/- mice made smaller granulomatous responses than did wild-type animals. These data clearly show that IL-4 is not necessary for the hepatic granuloma formation which occurs during experimental schistosomiasis.

Role of T-cell derived cytokines in the downregulation of immune responses in parasitic and retroviral infection.

Parasitic infection is frequently accompanied by a downregulation in host cell-mediated immunity. Recent studies suggest that this modulation of helper T cells and effector cell function can at least in part be attributed to the action of a set of inhibitory cytokines produced by T lymphocytes as well as by a number of other cell types. The best characterized of these inhibitory lymphokines are IL-4, IL-10 and TGF-beta. Interestingly, both IL-4 and IL-10 are produced by the Th2 but not the Th1 subset of CD4+ helper cells. The former subset dominates in many situations of chronic or exacerbated parasitic infection and is thought to suppress Th1 function as a consequence of the cross-regulatory activity of these two cytokines. The latter hypothesis is supported by recent experiments demonstrating that mAb-mediated neutralization of IL-10 reverses suppressed IFN-gamma responses and/or disease susceptibility in mice with parasitic infections. In vivo neutralization of TGF-beta has also been reported to increase host resistance to parasite challenge. In addition to suppressing T-cell differentiation, function or proliferation, IL-4, IL-10 and TGF-beta each inhibit the ability of IFN-gamma to activate macrophages for killing of both intracellular and extracellular parasites. Moreover, the three cytokines are able to synergize with each other in downregulating these parasiticidal effects. Interestingly, each of the cytokines inhibits the production of reactive nitrogen oxides, an effector mechanism previously demonstrated to play a major role in parasite killing by activated macrophages. In the case of IL-10, this suppression of nitrogen oxide production appears to result from an inhibition of TNF-alpha synthesis leading to defective macrophage stimulation. While distant from parasites in their biology and phylogeny, some retroviruses also appear to induce an over-production in downregulatory cytokines which is closely associated with the onset of immunodeficiency. Thus, in an animal model involving infection of mice with LP-BM5 MuLV and in human HIV infection, Th2 (IL-10 and/or IL-4) cytokine synthesis is increased while Th1 (IFN-gamma and/or IL-2) cytokine production is suppressed. These observations suggest that cytokine-mediated cross-regulation may play a role in the pathogenesis of acquired immune deficiency disease, contributing both to the progression of retroviral infection and the increase in susceptibility to opportunistic infections and malignancy. Observations of similar cytokine cross-regulatory activities in organisms as diverse as helminths, protozoa and retroviruses predict that comparable mechanisms may operate in a wide variety of infectious diseases.

Regulation and biological function of helminth-induced cytokine responses.

The immunological hallmarks of infection with parasitic helminths, namely eosinophilia, mastocytosis and increased IgE synthesis, all appear to be induced by cytokines from the TH2 subset of CD4+ T cells: IgE production is stimulated by interleukin 4 (IL-4), eosinophilia by IL-5 and mastocytosis by IL-3 and IL-4. Here, Fred Finkelman and colleagues argue that the functional significance of the eosinophilia-mastocytosis-IgE axis in helminth infection is unclear and suggest that in some worm infections TH2-cell cytokines may contribute to host protection, while in others they may promote parasite survival.

Induction of protective immunity against Schistosoma mansoni by vaccination with schistosome paramyosin (Sm97), a nonsurface parasite antigen.

Paramyosin (Sm97), a 97-kDa myofibrillar protein identified by the unusually monospecific antibody response induced by intradermal vaccination of mice with a complex soluble worm antigen preparation (SWAP) of adult Schistosoma mansoni administered with bacillus Calmette-Guérin (BCG), was purified and tested for its capacity to protect mice against challenge infection. When administered intradermally with BCG at total doses of only 4-40 micrograms per mouse, both the native molecule and a recombinant expression product containing approximately 50% of the whole protein were found to confer significant resistance (26-33%) against challenge infection, while 2 mg of unfractionated SWAP was required to induce similar levels of protection. In addition, paramyosin was shown to stimulate T lymphocytes from vaccinated mice to produce lymphokines [e.g., gamma interferon (IFN-gamma)] that activate macrophages to kill schistosomula. Neither schistosome myosin nor a heterologous paramyosin from a different invertebrate genus were protective, indicating a requirement for specific epitopes in the immunization. That the protection induced by paramyosin involves a T-cell-mediated mechanism was supported by the failure of anti-paramyosin antibodies to passively transfer significant resistance to infection to recipient mice. Lymphocytes from mice vaccinated with paramyosin were found to produce IFN-gamma in response to living schistosomula, suggesting that during challenge infection of vaccinated hosts, paramyosin (a nonsurface antigen) may elicit a protective T-cell response as a consequence of its release from migrating parasite larvae. Paramyosin-depleted SWAP was also found to be protective as well as stimulatory for T lymphocytes from SWAP-vaccinated mice, indicating that other antigens in this preparation may have immunoprophylactic potential. In summary, these results (i) suggest that the induction of T-cell-dependent cell-mediated immunity against soluble nonsurface antigens may be an effective strategy for immunization against multicellular parasites and (ii) in the case of schistosomes, identify paramyosin as a candidate vaccine immunogen in this category.