Induction of antigen specific intrahepatic CD8+ T cell responses by a secreted heat shock protein based gp96-Ig-PfCA malaria vaccine.

Introduction: A highly efficacious and durable vaccine against malaria is an essential tool for global malaria eradication. One of the promising strategies to develop such a vaccine is to induce robust CD8+ T cell mediated immunity against malaria liver-stage parasites. Methods: Here we describe a novel malaria vaccine platform based on a secreted form of the heat shock protein, gp96-immunoglobulin, (gp96-Ig) to induce malaria antigen specific, memory CD8+ T cells. Gp96-Ig acts as an adjuvant to activate antigen presenting cells (APCs) and chaperone peptides/antigens to APCs for cross presentation to CD8+ T cells. Results: Our study shows that vaccination of mice and rhesus monkeys with HEK-293 cells transfected with gp96-Ig and two well-known Plasmodium falciparum CSP and AMA1 (PfCA) vaccine candidate antigens, induces liver-infiltrating, antigen specific, memory CD8+ T cell responses. The majority of the intrahepatic CSP and AMA1 specific CD8+ T cells expressed CD69 and CXCR3, the hallmark of tissue resident memory T cells (Trm). Also, we found intrahepatic, antigen-specific memory CD8+ T cells secreting IL-2, which is relevant for maintenance of effective memory responses in the liver. Discussion: Our novel gp96-Ig malaria vaccine strategy represents a unique approach to induce liver-homing, antigen-specific CD8+ T cells critical for Plasmodium liver-stage protection.

Exposure-related, global alterations in innate and adaptive immunity; a consideration for re-use of non-human primates in research.

BACKGROUND: Non-human primates (NHPs) play an important role in biomedical research, where they are often being re-used in multiple research studies over the course of their life-time. Researchers employ various study-specific screening criteria to reduce potential variables associated with subsequent re-use of NHPs. However, criteria set for NHP re-assignments largely neglect the impact of previous exposures on overall biology. Since the immune system is a key determinant of overall biological outcome, an altered biological state could be predicted by monitoring global changes in the immune profile. We postulate that every different exposure or a condition can generate a unique global immune profile in NHPs. METHODS: Changes in the global immune profile were evaluated in three different groups of rhesus macaques previously enrolled in dengue or malaria vaccine studies over six months after their last exposure. Naïve animals served as the baseline. Fresh blood samples were stained with various immune cell surface markers and analyzed by multi-color flow-cytometry to study immune cell dynamics in the peripheral blood. Serum cytokine profile in the pre-exposed animals were analyzed by mesoscale assay using a customized U-PLEX NHP biomarker panel of 12 cytokines/chemokines. RESULTS: Pre-exposed macaques showed altered dynamics in circulating cytokines and certain innate and adaptive immune cell subsets such as monocytes, HLA-DR+NKT cells, B cells and T cells. Some of these changes were transient, while some lasted for more than six months. Each group seemed to develop a global immune profile unique to their particular exposure. CONCLUSION: Our data strongly suggest that re-used NHPs should be evaluated for long-term, overall immunological changes and randomly assigned to new studies to avoid study bias.

Humanized DRAGA mice immunized with Plasmodium falciparum sporozoites and chloroquine elicit protective pre-erythrocytic immunity.

BACKGROUND: Human-immune-system humanized mouse models can bridge the gap between humans and conventional mice for testing human vaccines. The HLA-expressing humanized DRAGA (HLA-A2.HLA-DR4.Rag1KO.IL2RγcKO.NOD) mice reconstitute a functional human-immune-system and sustain the complete life cycle of Plasmodium falciparum. Herein, the DRAGA mice were investigated for immune responses following immunization with live P. falciparum sporozoites under chloroquine chemoprophylaxis (CPS-CQ), an immunization approach that showed in human trials to confer pre-erythrocytic immunity. RESULTS: The CPS-CQ immunized DRAGA mice (i) elicited human CD4 and CD8 T cell responses to antigens expressed by P. falciparum sporozoites (Pfspz) and by the infected-red blood cells (iRBC). The Pfspz-specific human T cell responses were found to be systemic (spleen and liver), whereas the iRBCs-specific human T cell responses were more localized to the liver, (ii) elicited stronger antibody responses to the Pfspz than to the iRBCs, and (iii) they were protected against challenge with infectious Pfspz but not against challenge with iRBCs. CONCLUSIONS: The DRAGA mice represent a new pre-clinical model to investigate the immunogenicity and protective efficacy of P. falciparum malaria vaccine candidates.

Differential effect of HLA class-I versus class-II transgenes on human T and B cell reconstitution and function in NRG mice.

Humanized mice expressing Human Leukocyte Antigen (HLA) class I or II transgenes have been generated, but the role of class I vs class II on human T and B cell reconstitution and function has not been investigated in detail. Herein we show that NRG (NOD.RagKO.IL2RγcKO) mice expressing HLA-DR4 molecules (DRAG mice) and those co-expressing HLA-DR4 and HLA-A2 molecules (DRAGA mice) did not differ in their ability to develop human T and B cells, to reconstitute cytokine-secreting CD4 T and CD8 T cells, or to undergo immunoglobulin class switching. In contrast, NRG mice expressing only HLA-A2 molecules (A2 mice) reconstituted lower numbers of CD4 T cells but similar numbers of CD8 T cells. The T cells from A2 mice were deficient at secreting cytokines, and their B cells could not undergo immunoglobulin class switching. The inability of A2 mice to undergo immunoglobulin class switching is due to deficient CD4 helper T cell function. Upon immunization, the frequency and cytotoxicity of antigen-specific CD8 T cells in DRAGA mice was significantly higher than in A2 mice. The results indicated a multifactorial effect of the HLA-DR4 transgene on development and function of human CD4 T cells, antigen-specific human CD8 T cells, and immunoglobulin class switching.

TFH cells accumulate in mucosal tissues of humanized-DRAG mice and are highly permissive to HIV-1.

CD4(+) T follicular helper cells (TFH) in germinal centers are required for maturation of B-cells. While the role of TFH-cells has been studied in blood and lymph nodes of HIV-1 infected individuals, its role in the mucosal tissues has not been investigated. We show that the gut and female reproductive tract (FRT) of humanized DRAG mice have a high level of human lymphocytes and a high frequency of TFH (CXCR5(+)PD-1(++)) and precursor-TFH (CXCR5(+)PD-1(+)) cells. The majority of TFH-cells expressed CCR5 and CXCR3 and are the most permissive to HIV-1 infection. A single low-dose intravaginal HIV-1 challenge of humanized DRAG mice results in 100% infectivity with accumulation of TFH-cells mainly in the Peyer's patches and FRT. The novel finding of TFH-cells in the FRT may contribute to the high susceptibility of DRAG mice to HIV-1 infection. This mouse model thus provides new opportunities to study TFH-cells and to evaluate HIV-1 vaccines.

Humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice sustain the complex vertebrate life cycle of Plasmodium falciparum malaria.

BACKGROUND: Malaria is a deadly infectious disease affecting millions of people in tropical and sub-tropical countries. Among the five species of Plasmodium parasites that infect humans, Plasmodium falciparum accounts for the highest morbidity and mortality associated with malaria. Since humans are the only natural hosts for P. falciparum, the lack of convenient animal models has hindered the understanding of disease pathogenesis and prompted the need of testing anti-malarial drugs and vaccines directly in human trials. Humanized mice hosting human cells represent new pre-clinical models for infectious diseases that affect only humans. In this study, the ability of human-immune-system humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice to sustain infection with P. falciparum was explored. METHODS: Four week-old DRAG mice were infused with HLA-matched human haematopoietic stem cells (HSC) and examined for reconstitution of human liver cells and erythrocytes. Upon challenge with infectious P. falciparum sporozoites (NF54 strain) humanized DRAG mice were examined for liver stage infection, blood stage infection, and transmission to Anopheles stephensi mosquitoes. RESULTS: Humanized DRAG mice reconstituted human hepatocytes, Kupffer cells, liver endothelial cells, and erythrocytes. Upon intravenous challenge with P. falciparum sporozoites, DRAG mice sustained liver to blood stage infection (average 3-5 parasites/microlitre blood) and allowed transmission to An. stephensi mosquitoes. Infected DRAG mice elicited antibody and cellular responses to the blood stage parasites and self-cured the infection by day 45 post-challenge. CONCLUSIONS: DRAG mice represent the first human-immune-system humanized mouse model that sustains the complex vertebrate life cycle of P. falciparum without the need of exogenous injection of human hepatocytes/erythrocytes or P. falciparum parasite adaptation. The ability of DRAG mice to elicit specific human immune responses to P. falciparum parasites may help deciphering immune correlates of protection and to identify protective malaria antigens.

Humanized HLA-DR4 mice fed with the protozoan pathogen of oysters Perkinsus marinus (Dermo) do not develop noticeable pathology but elicit systemic immunity.

Perkinsus marinus (Phylum Perkinsozoa) is a marine protozoan parasite responsible for "Dermo" disease in oysters, which has caused extensive damage to the shellfish industry and estuarine environment. The infection prevalence has been estimated in some areas to be as high as 100%, often causing death of infected oysters within 1-2 years post-infection. Human consumption of the parasites via infected oysters is thus likely to occur, but to our knowledge the effect of oral consumption of P. marinus has not been investigated in humans or other mammals. To address the question we used humanized mice expressing HLA-DR4 molecules and lacking expression of mouse MHC-class II molecules (DR4.EA(0)) in such a way that CD4 T cell responses are solely restricted by the human HLA-DR4 molecule. The DR4.EA(0) mice did not develop diarrhea or any detectable pathology in the gastrointestinal tract or lungs following single or repeated feedings with live P. marinus parasites. Furthermore, lymphocyte populations in the gut associated lymphoid tissue and spleen were unaltered in the parasite-fed mice ruling out local or systemic inflammation. Notably, naïve DR4.EA(0) mice had antibodies (IgM and IgG) reacting against P. marinus parasites whereas parasite specific T cell responses were undetectable. Feeding with P. marinus boosted the antibody responses and stimulated specific cellular (IFNγ) immunity to the oyster parasite. Our data indicate the ability of P. marinus parasites to induce systemic immunity in DR4.EA(0) mice without causing noticeable pathology, and support rationale grounds for using genetically engineered P. marinus as a new oral vaccine platform to induce systemic immunity against infectious agents.

HLA class II (DR0401) molecules induce Foxp3+ regulatory T cell suppression of B cells in Plasmodium yoelii strain 17XNL malaria.

Unlike human malaria parasites that induce persistent infection, some rodent malaria parasites, like Plasmodium yoelii strain 17XNL (Py17XNL), induce a transient (self-curing) malaria infection. Cooperation between CD4 T cells and B cells to produce antibodies is thought to be critical for clearance of Py17XNL parasites from the blood, with major histocompatibility complex (MHC) class II molecules being required for activation of CD4 T cells. In order to better understand the correspondence between murine malaria models and human malaria, and in particular the role of MHC (HLA) class II molecules, we studied the ability of humanized mice expressing human HLA class II molecules to clear Py17XNL infection. We showed that humanized mice expressing HLA-DR4 (DR0401) molecules and lacking mouse MHC class II molecules (EA(0)) have impaired production of specific antibodies to Py17XNL and cannot cure the infection. In contrast, mice expressing HLA-DR4 (DR0402), HLA-DQ6 (DQ0601), HLA-DQ8 (DQ0302), or HLA-DR3 (DR0301) molecules in an EA(0) background were able to elicit specific antibodies and self-cure the infection. In a series of experiments, we determined that the inability of humanized DR0401.EA(0) mice to elicit specific antibodies was due to expansion and activation of regulatory CD4(+) Foxp3(+) T cells (Tregs) that suppressed B cells to secrete antibodies through cell-cell interactions. Treg depletion allowed the DR0401.EA(0) mice to elicit specific antibodies and self-cure the infection. Our results demonstrated a differential role of MHC (HLA) class II molecules in supporting antibody responses to Py17XNL malaria and revealed a new mechanism by which malaria parasites stimulate B cell-suppressogenic Tregs that prevent clearance of infection.

Strain-specific protective effect of the immunity induced by live malarial sporozoites under chloroquine cover.

The efficacy of a whole-sporozoite malaria vaccine would partly be determined by the strain-specificity of the protective responses against malarial sporozoites and liver-stage parasites. Evidence from previous reports were inconsistent, where some studies have shown that the protective immunity induced by irradiated or live sporozoites in rodents or humans were cross-protective and in others strain-specific. In the present work, we have studied the strain-specificity of live sporozoite-induced immunity using two genetically and immunologically different strains of Plasmodium cynomolgi, Pc746 and PcCeylon, in toque monkeys. Two groups of monkeys were immunized against live sporozoites of either the Pc746 (n = 5), or the PcCeylon (n = 4) strain, by the bites of 2-4 sporozoite-infected Anopheles tessellates mosquitoes per monkey under concurrent treatments with chloroquine and primaquine to abrogate detectable blood infections. Subsequently, a group of non-immunized monkeys (n = 4), and the two groups of immunized monkeys were challenged with a mixture of sporozoites of the two strains by the bites of 2-5 infective mosquitoes from each strain per monkey. In order to determine the strain-specificity of the protective immunity, the proportions of parasites of the two strains in the challenge infections were quantified using an allele quantification assay, Pyrosequencing™, based on a single nucleotide polymorphism (SNP) in the parasites' circumsporozoite protein gene. The Pyrosequencing™ data showed that a significant reduction of parasites of the immunizing strain in each group of strain-specifically immunized monkeys had occurred, indicating a stronger killing effect on parasites of the immunizing strain. Thus, the protective immunity developed following a single, live sporozoite/chloroquine immunization, acted specifically against the immunizing strain and was, therefore, strain-specific. As our experiment does not allow us to determine the parasite stage at which the strain-specific protective immunity is directed, it is possible that the target of this immunity could be either the pre-erythrocytic stage, or the blood-stage, or both.