T cells as mediators of protective immunity against liver stages of Plasmodium

T cells from different subsets play a major role in protective immunity against pre-erythrocytic stages of malaria parasites. Exposure of humans and animals to malaria sporozoites induces (alphabeta CD8(+) and CD4(+) T cells specific for antigens expressed in pre-erythrocytic stages of Plasmodium. These T cells inhibit parasite development in the liver, and immunization with subunit vaccines expressing the respective antigenic moieties confers protection against sporozoite challenge. gammadelta and natural killer T cells can also play a role in protective immunity. Recent studies with mice transgenic for the alphabeta T-cell receptor have revealed the existence of complex mechanisms regulating the induction and development of these responses.     

Protective T cell immunity against malaria liver stage after vaccination with live sporozoites under chloroquine treatment

In this study we present the first systematic analysis of the immunity induced by normal Plasmodium yoelii sporozoites in mice. Immunization with sporozoites, which was conducted under chloroquine treatment to minimize the influence of blood stage parasites, induced a strong protection against a subsequent sporozoite and, to a lesser extent, against infected RBC challenges. The protection induced by this immunization protocol proved to be very effective. Induction of this protective immunity depended on the presence of liver stage parasites, as primaquine treatment concurrent with sporozoite immunization abrogated protection. Protection was not found to be mediated by the Abs elicited against pre-erythrocytic and blood stage parasites, as demonstrated by inhibition assays of sporozoite penetration or development in vitro and in vivo assays of sporozoite infectivity or blood stage parasite development. CD4(+) and CD8(+) T cells were, however, responsible for the protection through the induction of IFN-gamma and NO.     

Protective immunity induced with malaria vaccine, RTS,S, is linked to Plasmodium falciparum circumsporozoite protein-specific CD4+ and CD8+ T cells producing IFN-gamma

The Plasmodium falciparum circumsporozoite (CS) protein-based pre-erythrocytic stage vaccine, RTS,S, induces a high level of protection against experimental sporozoite challenge. The immune mechanisms that constitute protection are only partially understood, but are presumed to rely on Abs and T cell responses. In the present study we compared CS protein peptide-recalled IFN-gamma reactivity of pre- and RTS,S-immune lymphocytes from 20 subjects vaccinated with RTS,S. We observed elevated IFN-gamma in subjects protected by RTS,S; moreover, both CD4(+) and CD8(+) T cells produced IFN-gamma in response to CS protein peptides. Significantly, protracted protection, albeit observed only in two of seven subjects, was associated with sustained IFN-gamma response. This is the first study demonstrating correlation in a controlled Plasmodia sporozoite challenge study between protection induced by a recombinant malaria vaccine and Ag-specific T cell responses. Field-based malaria vaccine studies are in progress to validate the establishment of this cellular response as a possible in vitro correlate of protective immunity to exo-erythrocytic stage malaria vaccines.     

CD8+ cellular immunity mediates rAd5 vaccine protection against Ebola virus infection of nonhuman primates

Vaccine-induced immunity to Ebola virus infection in nonhuman primates (NHPs) is marked by potent antigen-specific cellular and humoral immune responses; however, the immune mechanism of protection remains unknown. Here we define the immune basis of protection conferred by a highly protective recombinant adenovirus virus serotype 5 (rAd5) encoding Ebola virus glycoprotein (GP) in NHPs. Passive transfer of high-titer polyclonal antibodies from vaccinated Ebola virus-immune cynomolgus macaques to naive macaques failed to confer protection against disease, suggesting a limited role of humoral immunity. In contrast, depletion of CD3(+) T cells in vivo after vaccination and immediately before challenge eliminated immunity in two vaccinated macaques, indicating a crucial requirement for T cells in this setting. The protective effect was mediated largely by CD8(+) cells, as depletion of CD8(+) cells in vivo using the cM-T807 monoclonal antibody (mAb), which does not affect CD4(+) T cell or humoral immune responses, abrogated protection in four out of five subjects. These findings indicate that CD8(+) cells have a major role in rAd5-GP-induced immune protection against Ebola virus infection in NHPs. Understanding the immunologic mechanism of Ebola virus protection will facilitate the development of vaccines for Ebola and related hemorrhagic fever viruses in humans.     

Plasmodium berghei-infected primary hepatocytes process and present the circumsporozoite protein to specific CD8+ T cells in vitro

A substantial and protective response against malaria liver stages is directed against the circumsporozoite protein (CSP) and involves induction of CD8(+) T cells and production of IFN-gamma. CSP-derived peptides have been shown to be presented on the surface of infected hepatocytes in the context of MHC class I molecules. However, little is known about how the CSP and other sporozoite Ags are processed and presented to CD8(+) T cells. We investigated how primary hepatocytes from BALB/c mice process the CSP of Plasmodium berghei after live sporozoite infection and present CSP-derived peptides to specific H-2K(d)-restricted CD8(+) T cells in vitro. Using both wild-type and spect(-/-) P. berghei sporozoites, we show that both infected and traversed primary hepatocytes process and present the CSP. The processing and presentation pathway was found to involve the proteasome, Ag transport through a postendoplasmic reticulum compartment, and aspartic proteases. Thus, it can be hypothesized that infected hepatocytes can contribute in vivo to the elicitation and expansion of a T cell response.     

Vaccine protection against simian immunodeficiency virus in monkeys using recombinant gamma-2 herpesvirus

Recombinant strains of replication-competent rhesus monkey rhadinovirus (RRV) were constructed in which strong promoter/enhancer elements were used to drive expression of simian immunodeficiency virus (SIV) Env or Gag or a Rev-Tat-Nef fusion protein. Cultured rhesus monkey fibroblasts infected with each recombinant strain were shown to express the expected protein. Three RRV-negative and two RRV-positive rhesus monkeys were inoculated intravenously with a mixture of these three recombinant RRVs. Expression of SIV Gag was readily detected in lymph node biopsy specimens taken at 3 weeks postimmunization. Impressive anti-SIV cellular immune responses were elicited on the basis of major histocompatibility complex (MHC) tetramer staining and gamma interferon enzyme-linked immunospot (ELISPOT) assays. Responses were much greater in magnitude in the monkeys that were initially RRV negative but were still readily detected in the two monkeys that were naturally infected with RRV at the time of immunization. By 3 weeks postimmunization, responses measured by MHC tetramer staining in the two Mamu-A*01(+) RRV-negative monkeys reached 9.3% and 13.1% of all CD8(+) T cells in peripheral blood to the Gag CM9 epitope and 2.3% and 7.3% of all CD8(+) T cells in peripheral blood to the Tat SL8 epitope. Virus-specific CD8(+) T cell responses persisted at high levels up to the time of challenge at 18 weeks postimmunization, and responding cells maintained an effector memory phenotype. Despite the ability of the RRVenv recombinant to express high levels of Env in cultured cells, and despite the appearance of strong anti-RRV antibody responses in immunized monkeys, anti-Env antibody responses were below our ability to detect them. Immunized monkeys, together with three unimmunized controls, were challenged intravenously with 10 monkey infectious doses of SIVmac239. All five immunized monkeys and all three controls became infected with SIV, but peak viral loads were 1.2 to 3.0 log(10) units lower and chronic-phase viral loads were 1.0 to 3.0 log(10) units lower in immunized animals than the geometric mean of unimmunized controls. These differences were statistically significant. Anti-Env antibody responses following challenge indicated an anamnestic response in the vaccinated monkeys. These findings further demonstrate the potential of recombinant herpesviruses as preventive vaccines for AIDS. We hypothesize that this live, replication-competent, persistent herpesvirus vector could match, or come close to matching, live attenuated strains of SIV in the degree of protection if the difficulty with elicitation of anti-Env antibody responses can be overcome.     

CD8+ T effector memory cells protect against liver-stage malaria

Identification of correlates of protection for infectious diseases including malaria is a major challenge and has become one of the main obstacles in developing effective vaccines. We investigated protection against liver-stage malaria conferred by vaccination with adenoviral (Ad) and modified vaccinia Ankara (MVA) vectors expressing pre-erythrocytic malaria Ags. By classifying CD8(+) T cells into effector, effector memory (T(EM)), and central memory subsets using CD62L and CD127 markers, we found striking differences in T cell memory generation. Although MVA induced accelerated central memory T cell generation, which could be efficiently boosted by subsequent Ad administration, it failed to protect against malaria. In contrast, Ad vectors, which permit persistent Ag delivery, elicit a prolonged effector T cell and T(EM) response that requires long intervals for an efficient boost. A preferential T(EM) phenotype was maintained in liver, blood, and spleen after Ad/MVA prime-boost regimens, and animals were protected against malaria sporozoite challenge. Blood CD8(+) T(EM) cells correlated with protection against malaria liver-stage infection, assessed by estimation of number of parasites emerging from the liver into the blood. The protective ability of Ag-specific T(EM) cells was confirmed by transfer experiments into naive recipient mice. Thus, we identify persistent CD8 T(EM) populations as essential for vaccine-induced pre-erythrocytic protection against malaria, a finding that has important implications for vaccine design.     

Global dysfunction of CD4 T-lymphocyte cytokine expression in simian-human immunodeficiency virus/SIV-infected monkeys is prevented by vaccination

Human immunodeficiency virus type 1 infection results in a dysfunction of CD4(+) T lymphocytes. The intracellular events contributing to that CD4(+) T-lymphocyte dysfunction remain incompletely elucidated, and it is unclear whether aspects of that dysfunction can be prevented. The present studies were pursued in a rhesus monkey model of AIDS to explore these issues. Loss of the capacity of peripheral blood CD4(+) T lymphocytes to express cytokines was first detected in simian immunodeficiency virus-infected monkeys during the peak of viral replication during primary infection and persisted thereafter. Moreover, infected monkeys with progressive disease had peripheral blood CD4(+) T lymphocytes that expressed significantly less cytokine than infected monkeys that had undetectable viral loads and intact CD4(+) T-lymphocyte counts. Importantly, CD4(+) T lymphocytes from vaccinated monkeys that effectively controlled the replication of a highly pathogenic immunodeficiency virus isolate following a challenge had a preserved functional capacity. These observations suggest that an intact cytokine expression capacity of CD4(+) T lymphocytes is associated with stable clinical status and that effective vaccines can mitigate against CD4(+) T-lymphocyte dysfunction following an AIDS virus infection.     

The dendritic cell-specific chemokine,dendritic cell-derived CC chemokine 1, enhances protective cell-mediated immunity to murine malaria

Cell-mediated immunity plays a crucial role in the control of many infectious diseases, necessitating the need for adjuvants that can augment cellular immune responses elicited by vaccines. It is well established that protection against one such disease, malaria, requires strong CD8(+) T cell responses targeted against the liver stages of the causative agent, Plasmodium spp. In this report we show that the dendritic cell-specific chemokine, dendritic cell-derived CC chemokine 1 (DC-CK1), which is produced in humans and acts on naive lymphocytes, can enhance Ag-specific CD8(+) T cell responses when coadministered with either irradiated Plasmodium yoelii sporozoites or a recombinant adenovirus expressing the P. yoelii circumsporozoite protein in mice. We further show that these enhanced T cell responses result in increased protection to malaria in immunized mice challenged with live P. yoelii sporozoites, revealing an adjuvant activity for DC-CK1. DC-CK1 appears to act preferentially on naive mouse lymphocytes, and its adjuvant effect requires IL-12, but not IFN-gamma or CD40. Overall, our results show for the first time an in vivo role for DC-CK1 in the establishment of primary T cell responses and indicate the potential of this chemokine as an adjuvant for vaccines against malaria as well as other diseases in which cellular immune responses are important.     

Cutting edge: attrition of Plasmodium-specific memory CD8 T cells results in decreased protection that is rescued by booster immunization

Sterile protection against infection with Plasmodium sporozoites requires high numbers of memory CD8 T cells. However, infections with unrelated pathogens, as may occur in areas endemic to malaria, can dramatically decrease pre-existing memory CD8 T cells. It remains unknown whether unrelated infections will compromise numbers of Plasmodium-specific memory CD8 T cells and thus limit the duration of antimalarial immunity generated by subunit vaccination. We show that P. berghei circumsporozoite-specific memory CD8 T cells underwent significant attrition in numbers in mice subjected to unrelated infections. Attrition was associated with preferential loss of effector memory CD8 T cells and reduced immunity to P. berghei sporozoite challenge. However, and of relevance to deployment of Plasmodium vaccines in areas endemic to malaria, attrition of memory CD8 T cells was reversed by booster immunization, which restored protection. These data suggest that regular booster immunizations may be required to sustain protective vaccine-induced Plasmodium-specific memory CD8 T cells in the face of attrition caused by unrelated infections.     

Immunity to the liver stage of malaria

The search for subunit vaccines against malaria has concentrated on asexual and sexual blood stage and sporozoite antigens. In recent years the search for the basis of the protection against sporozoite challenge obtained in mice immunized with irradiated sporozoites has focused attention on the liver or exoerythrocytic (EE) stage of the malaria life cycle. Here, Andreas Suhrbier looks at the various immune responses that appear to be active against this stage, which was once thought to be immunologically insignificant. The liver stage of malaria has thus emerged as a legitimate target for vaccine development.     

Protective CD8+ T cell responses against the pre-erythrocytic stages of malaria parasites: an overview

CD8+ T cells have been implicated as critical effector cells in protection against the pre-erythrocytic stage of malaria in mice and humans following irradiated sporozoite immunization. Immunization experiments in animal models by several investigators have suggested different strategies for vaccination against malaria and many of the targets from liver stage malaria antigens have been shown to be immunogenic and to protect mice from the sporozoite challenge. Several prime/boost protocols with replicating vectors, such as vaccinia/influenza, with non-replicating vectors, such as recombinant particles derived from yeast transposon (Ty-particles) and modified vaccinia virus Ankara, and DNA, significantly enhanced CD8+ T cell immunogenicity and also the protective efficacy against the circumsporosoite protein of Plasmodium berghei and P. yeti. Based on these experimental results the development of a CD8+ T cell inducing vaccine has moved forward from epitope identification to planning stages of safety and immunogenicity trials of candidate vaccines.     

Protection from experimental cerebral malaria with a single dose of radiation-attenuated, blood-stage Plasmodium berghei parasites

Background

Whole malaria parasites are highly effective in inducing immunity against malaria. Due to the limited success of subunit based vaccines in clinical studies, there has been a renewed interest in whole parasite-based malaria vaccines. Apart from attenuated sporozoites, there have also been efforts to use live asexual stage parasites as vaccine immunogens.

Methodology and Results

We used radiation exposure to attenuate the highly virulent asexual blood stages of the murine malaria parasite P. berghei to a non-replicable, avirulent form. We tested the ability of the attenuated blood stage parasites to induce immunity to parasitemia and the symptoms of severe malaria disease. Depending on the mouse genetic background, a single high dose immunization without adjuvant protected mice from parasitemia and severe disease (CD1 mice) or from experimental cerebral malaria (ECM) (C57BL/6 mice). A low dose immunization did not protect against parasitemia or severe disease in either model after one or two immunizations. The protection from ECM was associated with a parasite specific antibody response and also with a lower level of splenic parasite-specific IFN-γ production, which is a mediator of ECM pathology in C57BL/6 mice. Surprisingly, there was no difference in the sequestration of CD8+ T cells and CD45+ CD11b+ macrophages in the brains of immunized, ECM-protected mice.

Conclusions

This report further demonstrates the effectiveness of a whole parasite blood-stage vaccine in inducing immunity to malaria and explicitly demonstrates its effectiveness against ECM, the most pathogenic consequence of malaria infection. This experimental model will be important to explore the formulation of whole parasite blood-stage vaccines against malaria and to investigate the immune mechanisms that mediate protection against parasitemia and cerebral malaria.     

Human CD4+ T cells induced by synthetic peptide malaria vaccine are comparable to cells elicited by attenuated Plasmodium falciparum sporozoites

Peptide vaccines containing minimal epitopes of protective Ags provide the advantages of low cost, safety, and stability while focusing host responses on relevant targets of protective immunity. However, the limited complexity of malaria peptide vaccines raises questions regarding their equivalence to immune responses elicited by the irradiated sporozoite vaccine, the "gold standard" for protective immunity. A panel of CD4+ T cell clones was derived from volunteers immunized with a peptide vaccine containing minimal T and B cell epitopes of the Plasmodium falciparum circumsporozoite protein to compare these with previously defined CD4+ T cell clones from volunteers immunized with irradiated P. falciparum sporozoites. As found following sporozoite immunization, the majority of clones from the peptide-immunized volunteers recognized the T* epitope, a predicted universal T cell epitope, in the context of multiple HLA DR and DQ molecules. Peptide-induced T cell clones were of the Th0 subset, secreting high levels of IFN-gamma as well as variable levels of Th2-type cytokines (IL-4, IL-6). The T* epitope overlaps a polymorphic region of the circumsporozoite protein and strain cross-reactivity of the peptide-induced clones correlated with recognition of core epitopes overlapping the conserved regions of the T* epitope. Importantly, as found following sporozoite immunization, long-lived CD4+ memory cells specific for the T* epitope were detectable 10 mo after peptide immunization. These studies demonstrate that malaria peptides containing minimal epitopes can elicit human CD4+ T cells with fine specificity and potential effector function comparable to those elicited by attenuated P. falciparum sporozoites.     

IL-12 and NK cells are required for antigen-specific adaptive immunity against malaria initiated by CD8+ T cells in the Plasmodium yoelii model.

CD8+ T cells have been implicated as critical effector cells in protection against preerythrocytic stage malaria, including the potent protective immunity of mice and humans induced by immunization with radiation-attenuated Plasmodium spp. sporozoites. This immunity is directed against the Plasmodium spp. parasite developing within the host hepatocyte and for a number of years has been presumed to be mediated directly by CD8+ CTL or indirectly by IFN-gamma released from CD8+ T cells. In this paper, in BALB/c mice, we establish that after immunization with irradiated sporozoites or DNA vaccines parasite-specific CD8+ T cells trigger a novel mechanism of adaptive immunity that is dependent on T cell- and non-T cell-derived cytokines, in particular IFN-gamma and IL-12, and requires NK cells but not CD4+ T cells. The absolute requirement for CD8+ T cells to initiate such an effector mechanism, and the requirement for IL-12 and NK cells in such vaccine-induced protective immunity, are unique and underscore the complexity of the immune responses that protect against malaria and other intracellular pathogens.     

A New Approach to AIDS Research and Prevention: The Use of Gene-Mutated HIV-1/SIV Chimeric Viruses for Anti-HIV-1 Live-Attenuated Vaccines

The lack of a suitable animal model is a major obstacle to developing anti-HIV-1 vaccines. We successfully generated an SIVmac/HIV-1 chimeric virus (SHIV) (designated as NM-3rN) that contains the HIV-1 env gene and is infectious to macaque monkeys. Challenging the vaccinated macaque monkeys with NM-3rN, we developed an evaluation system for anti-HIV-1 Env-targeted vaccines. For the purpose of making the vaccine, a series of gene-mutated SHIVs were constructed. The monkeys vaccinated with these SHIVs had long-term anti-virus immunities without manifesting the disease, and became resistant to a challenge inoculation with NM-3rN. The sera from a monkey showed that, after the vaccination, the neutralizing antibodies not only against the parental HIV-1 but also against an antigenically different HIV-1 were raised. In vivo experiments confirmed that the vaccinated monkeys were protected from the challenge inoculum of an antigenically different SHIV-MN. Vaccination of monkeys with the attenuated SHIVs showed that further gene-deletion of the SHIV resulted in less immunogenicity. Nevertheless, the attenuated SHIVs had a vaccine effect against the challenge inoculation. In addition to specific immunities including neutralizing antibodies and cytotoxic T cells, a more complicated immune mechanism induced by live vaccine appears to play a role in this protection. Our data suggest that the live vaccine can induce strong and wide-range immunity against HIV-1. These SHIVs should contribute to understanding the pathogenicity of AIDS and to the development of future anti-HIV-1 live vaccines for humans.     

Sterile Protection against Plasmodium knowlesi in Rhesus Monkeys from a Malaria Vaccine: Comparison of Heterologous Prime Boost Strategies

Using newer vaccine platforms which have been effective against malaria in rodent models, we tested five immunization regimens against Plasmodium knowlesi in rhesus monkeys. All vaccines included the same four P. knowlesi antigens: the pre-erythrocytic antigens CSP, SSP2, and erythrocytic antigens AMA1, MSP1. We used four vaccine platforms for prime or boost vaccinations: plasmids (DNA), alphavirus replicons (VRP), attenuated adenovirus serotype 5 (Ad), or attenuated poxvirus (Pox). These four platforms combined to produce five different prime/boost vaccine regimens: Pox alone, VRP/Pox, VRP/Ad, Ad/Pox, and DNA/Pox. Five rhesus monkeys were immunized with each regimen, and five Control monkeys received a mock vaccination. The time to complete vaccinations was 420 days. All monkeys were challenged twice with 100 P. knowlesi sporozoites given IV. The first challenge was given 12 days after the last vaccination, and the monkeys receiving the DNA/Pox vaccine were the best protected, with 3/5 monkeys sterilely protected and 1/5 monkeys that self-cured its parasitemia. There was no protection in monkeys that received Pox malaria vaccine alone without previous priming. The second sporozoite challenge was given 4 months after the first. All 4 monkeys that were protected in the first challenge developed malaria in the second challenge. DNA, VRP and Ad5 vaccines all primed monkeys for strong immune responses after the Pox boost. We discuss the high level but short duration of protection in this experiment and the possible benefits of the long interval between prime and boost.     

The species specificity of immunity generated by live whole organism immunisation with erythrocytic and pre-erythrocytic stages of rodent malaria parasites and implications for vaccine development

A promising strategy for the development of a malaria vaccine involves the use of attenuated whole parasites, as these present a greater repertoire of antigens to the immune system than subunit vaccines. The complexity of the malaria parasite's life cycle offers multiple stages on which to base an attenuated whole organism vaccine. An important consideration in the design and employment of such vaccines is the diversity of the parasites that are infective to humans. The most valuable vaccine would be one that was effective against multiple species/strains of malaria parasite. Here we compare the species specificity of pre-erythrocytic and erythrocytic whole organism vaccination using live parasites with anti-malarial drug attenuation. The cross-stage protection afforded by each vaccination strategy, and the possibility that immunity against one stage may be abrogated by exposure to other stages of both homologous and heterologous parasites was also assessed. The rodent malaria parasites Plasmodium yoelii yoelii and Plasmodium vinckei lentum are to address these questions, as they offer the widest possible genetic distance between sub-species of malaria parasites infectious to rodents. It was found that both erythrocytic and pre-erythrocytic stage immunity generated by live, attenuated parasite vaccination have species-specific components, with pre-erythrocytic stage immunity offering a much broader pan-species protection. We show that the protection achieved following sporozoite inoculation with concurrent mefloquine treatment is almost entirely dependent of CD8(+) T-cells. Evidence is presented for cross-stage protection between erythrocytic and pre-erythrocytic stage vaccination. Finally, it is shown that, with these species, an erythrocytic stage infection of either a homologous or heterologous species following immunisation with pre-erythrocytic stages does not abrogate this immunity. This is the first direct comparison of the specificity and efficacy of erythrocytic and pre-erythrocytic stage whole organism vaccination strategies utilising the same parasite species pair.     

Immune response to pre-erythrocytic stages of malaria parasites

Immunization with radiation-attenuated Plasmodium spp. sporozoites induces sterile protective immunity against parasite challenge. This immunity is targeted primarily against the intrahepatic parasite and appears to be sustained long term even in the absence of sporozoite exposure. It is mediated by multifactorial mechanisms, including T cells directed against parasite antigens expressed in the liver stage of the parasite life cycle and antibodies directed against sporozoite surface proteins. In rodent models, CD8+ T cells have been implicated as the principal effector cells, and IFN-gamma as a critical effector molecule. IL-4 secreting CD4+ T cells are required for induction of the CD8+ T cell responses, and Th1 CD4+ T cells provide help for optimal CD8+ T cell effector activity. Components of the innate immune system, including gamma-delta T cells, natural killer cells and natural killer T cells, also play a role. The precise nature of pre-erythrocytic stage immunity in humans, including the contribution of these immune responses to the age-dependent immunity naturally acquired by residents of malaria endemic areas, is still poorly defined. The importance of immune effector targets at the pre-erythrocytic stage of the parasite life cycle is highlighted by the fact that infection-blocking immunity in humans rarely, if ever, occurs under natural conditions. Herein, we review our current understanding of the molecular and cellular aspects of pre-erythrocytic stage immunity.     

Identification of novel HLA-DR1-restricted epitopes from the hepatitis B virus envelope protein in mice expressing HLA-DR1 and vaccinated human subjects

Helper T lymphocytes that control CD8(+) T-cell and antibody responses are key elements for the resolution of infection by the hepatitis B virus and for the development of effective immunological memory after hepatitis B vaccination. We have used H-2 class II-deficient mice that express the human MHC class II molecule, HLA-DR1, to identify novel hepatitis B virus envelope-derived T helper epitopes. We confirmed the immunogenicity of a previously described HLA-DR1-restricted epitope, and identified three novel epitopes. CD4(+) T-cell immune responses against these epitopes were detected in peripheral blood mononuclear cells from HLA-DR1(+) individuals vaccinated against hepatitis B. We showed that subjects receiving the currently available hepatitis B vaccines do not develop cross-reactive T helper responses against one of the novel epitopes which are structurally variable between different hepatitis B virus subtypes. These findings highlight the need for developing vaccines against a wider range of viral subtypes, and establish humanized mice as a convenient tool for identifying new immunogenic epitopes from pathogens.