IFNγ Responses to Pre-erythrocytic and Blood-stage Malaria Antigens Exhibit Differential Associations With Past Exposure and Subsequent Protection.

BACKGROUND: The malaria-specific T-cell response is believed to be important for protective immunity. Antimalarial chemoprevention may affect this response by altering exposure to malaria antigens. METHODS: We performed interferon γ (IFNγ) ELISpot assays to assess the cellular immune response to blood-stage and pre-erythrocytic antigens longitudinally from 1 to 3 years of age in 196 children enrolled in a randomized trial of antimalarial chemoprevention in Tororo, Uganda, an area of high transmission intensity. RESULTS: IFNγ responses to blood-stage antigens, particularly MSP1, were frequently detected, strongly associated with recent malaria exposure, and lower in those adherent to chemoprevention compared to nonadherent children and those randomized to no chemoprevention. IFNγ responses to pre-erythrocytic antigens were infrequent and similar between children randomized to chemoprevention or no chemoprevention. Responses to blood-stage antigens were not associated with subsequent protection from malaria (aHR 0.96, P = .83), but responses to pre-erythrocytic antigens were associated with protection after adjusting for prior malaria exposure (aHR 0.52, P = .009). CONCLUSIONS: In this high transmission setting, IFNγ responses to blood-stage antigens were common and associated with recent exposure to malaria but not protection from subsequent malaria. Responses to pre-erythrocytic antigens were uncommon, not associated with exposure but were associated with protection from subsequent malaria.

Full-length Plasmodium falciparum circumsporozoite protein administered with long-chain poly(I·C) or the Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion elicits potent antibody and CD4+ T cell immunity and protection in mice.

The Plasmodium falciparum circumsporozoite (CS) protein (CSP) is a major vaccine target for preventing malaria infection. Thus, developing strong and durable antibody and T cell responses against CSP with novel immunogens and potent adjuvants may improve upon the success of current approaches. Here, we compare four distinct full-length P. falciparum CS proteins expressed in Escherichia coli or Pichia pastoris for their ability to induce immunity and protection in mice when administered with long-chain poly(I · C) [poly(I · C)LC] as an adjuvant. CS proteins expressed in E. coli induced high-titer antibody responses against the NANP repeat region and potent CSP-specific CD4(+) T cell responses. Moreover, E. coli-derived CS proteins in combination with poly(I · C)LC induced potent multifunctional (interleukin 2-positive [IL-2(+)], tumor necrosis factor alpha-positive [TNF-α(+)], gamma interferon-positive [IFN-γ(+)]) CD4(+) effector T cell responses in blood, in spleen, and particularly in liver. Using transgenic Plasmodium berghei expressing the repeat region of P. falciparum CSP [Pb-CS(Pf)], we showed that there was a 1- to 4-log decrease in malaria rRNA in the liver following a high-dose challenge and ~50% sterilizing protection with a low-dose challenge compared to control levels. Protection was directly correlated with high-level antibody titers but not CD4(+) T cell responses. Finally, protective immunity was also induced using the Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) as the adjuvant, which also correlated with high antibody titers yet CD4(+) T cell immunity that was significantly less potent than that with poly(I · C)LC. Overall, these data suggest that full-length CS proteins and poly(I · C)LC or GLA-SE offer a simple vaccine formulation to be used alone or in combination with other vaccines for preventing malaria infection.

Identification and characterization of the host protein DNAJC14 as a broadly active flavivirus replication modulator.

Viruses in the Flavivirus genus of the Flaviviridae family are arthropod-transmitted and contribute to staggering numbers of human infections and significant deaths annually across the globe. To identify cellular factors with antiviral activity against flaviviruses, we screened a cDNA library using an iterative approach. We identified a mammalian Hsp40 chaperone protein (DNAJC14) that when overexpressed was able to mediate protection from yellow fever virus (YFV)-induced cell death. Further studies revealed that DNAJC14 inhibits YFV at the step of viral RNA replication. Since replication of bovine viral diarrhea virus (BVDV), a member of the related Pestivirus genus, is also known to be modulated by DNAJC14, we tested the effect of this host factor on diverse Flaviviridae family members. Flaviviruses, including the pathogenic Asibi strain of YFV, Kunjin, and tick-borne Langat virus, as well as a Hepacivirus, hepatitis C virus (HCV), all were inhibited by overexpression of DNAJC14. Mutagenesis showed that both the J-domain and the C-terminal domain, which mediates self-interaction, are required for anti-YFV activity. We found that DNAJC14 does not block YFV nor HCV NS2-3 cleavage, and using non-inhibitory mutants demonstrate that DNAJC14 is recruited to YFV replication complexes. Immunofluorescence analysis demonstrated that endogenous DNAJC14 rearranges during infection and is found in replication complexes identified by dsRNA staining. Interestingly, silencing of endogenous DNAJC14 results in impaired YFV replication suggesting a requirement for DNAJC14 in YFV replication complex assembly. Finally, the antiviral activity of overexpressed DNAJC14 occurs in a time- and dose-dependent manner. DNAJC14 overexpression may disrupt the proper stoichiometry resulting in inhibition, which can be overcome upon restoration of the optimal ratios due to the accumulation of viral nonstructural proteins. Our findings, together with previously published work, suggest that the members of the Flaviviridae family have evolved in unique and important ways to interact with this host Hsp40 chaperone molecule.

Evaluation of yellow fever virus 17D strain as a new vector for HIV-1 vaccine development.

The failure to develop an effective vaccine against HIV-1 infection has led the research community to seek new ways of raising qualitatively different antibody and cellular immune responses. Towards this goal, we investigated the yellow fever 17D vaccine strain (YF17D), one of the most effective vaccines ever made, as a platform for HIV-1 vaccine development. A test antigen, HIV-1 p24 (clade B consensus), was inserted near the 5' end of YF17D, in frame and upstream of the polyprotein (YF-5'/p24), or between the envelope and the first non-structural protein (YF-E/p24/NS1). In vitro characterization of these recombinants indicated that the gene insert was more stable in the context of YF-E/p24/NS1. This was confirmed in immunogenicity studies in mice. CD8(+) IFN-gamma T-cell responses against p24 were elicited by the YF17D recombinants, as were specific CD4(+) T cells expressing IFN-gamma and IL-2. A balanced CD4(+) and CD8(+) T-cell response was notable, as was the polyfunctionality of the responding cells. Finally, the protective efficacy of the YF17D recombinants, particularly YF-E/p24/NS1, in mice challenged with a vaccinia expressing HIV-1 Gag was demonstrated. These results suggest that YF17D warrants serious consideration as a live-attenuated vector for HIV-1 vaccine development.