Enhanced CD8+ T cell immune responses and protection elicited against Plasmodium berghei malaria by prime boost immunization regimens using a novel attenuated fowlpox virus

Sterile immunity can be provided against the pre-erythrocytic stages of malaria by IFN-gamma-secreting CD8(+) T cells that recognize parasite-infected hepatocytes. In this study, we have investigated the use of attenuated fowlpox virus (FPV) strains as recombinant vaccine vectors for eliciting CD8(+) T cells against Plasmodium berghei. The gene encoding the P. berghei circumsporozoite (PbCS) protein was inserted into an FPV vaccine strain licensed for use in chickens, Webster's FPV, and the novel FPV vaccine strain FP9 by homologous recombination. The novel FP9 strain proved more potent as a vaccine for eliciting CD8(+) T cell responses against the PbCS Ag. Sequential immunization with rFP9 and recombinant modified vaccinia virus Anakara (MVA) encoding the PbCS protein, administered by clinically acceptable routes, elicited potent CD8(+) T cell responses against the PbCS protein. This immunization regimen elicited substantial protection against a stringent liver-stage challenge with P. berghei and was more immunogenic and protective than DNA/MVA prime/boost immunization. However, further improvement was not achieved by sequential (triple) immunization with a DNA vaccine, FP9, and MVA.     

MVA-LACK as a safe and efficient vector for vaccination against leishmaniasis

An optimal vaccine against leishmaniasis should elicit parasite specific CD4+ and cytotoxic CD8+ T cells. In this investigation, we described a prime/boost immunization approach based on DNA and on poxvirus vectors (Western Reserve, WR, and the highly attenuated modified vaccinia virus Ankara, MVA), both expressing the LACK antigen of Leishmania infantum, that triggers different levels of specific CD8+ T cell responses and protection (reduction in lesion size and parasitemia) against L. major infection in mice. A prime/boost vaccination with DNA-LACK/MVA-LACK elicits higher CD8+ T cell responses than a similar protocol with the replication competent VV-LACK. Both CD4+ and CD8+ T cells were induced by DNA-LACK/MVA-LACK immunization. The levels of IFN-gamma and TNF-alpha secreting CD8+ T cells were higher in splenocytes from DNA-LACK/MVA-LACK than in DNA-LACK/VV-LACK immunized animals. Moreover, protection against L. major was significantly higher in DNA-LACK/MVA-LACK than in DNA-LACK/VV-LACK immunized animals when boosted with the same virus dose, and correlated with high levels of IFN-gamma and TNF-alpha secreting CD8+ T cells. In DNA-LACK/MVA-LACK vaccinated animals, the extent of lesion size reduction ranged from 65 to 92% and this protection was maintained for at least 17 weeks after challenge with the parasite. These findings demonstrate that in heterologous prime/boost immunization approaches, the protocol DNA-LACK/MVA-LACK is superior to DNA-LACK/VV-LACK in triggering specific CD8+ T cell immune responses and in conferring protection against cutaneous leishmaniasis. Thus, MVA-LACK is a safe and efficient vector for vaccination against leishmaniasis.     

Combining liver- and blood-stage malaria viral-vectored vaccines: investigating mechanisms of CD8+ T cell interference

Replication-deficient adenovirus and modified vaccinia virus Ankara (MVA) vectors expressing single pre-erythrocytic or blood-stage Plasmodium falciparum Ags have entered clinical testing using a heterologous prime-boost immunization approach. In this study, we investigated the utility of the same immunization regimen when combining viral vectored vaccines expressing the 42-kDa C terminus of the blood-stage Ag merozoite surface protein 1 and the pre-erythrocytic Ag circumsporozoite protein in the Plasmodium yoelii mouse model. We find that vaccine coadministration leads to maintained Ab responses and efficacy against blood-stage infection, but reduced secondary CD8(+) T cell responses against both Ags and efficacy against liver-stage infection. CD8(+) T cell interference can be minimized by coadministering the MVA vaccines at separate sites, resulting in enhanced liver-stage efficacy in mice immunized against both Ags compared with just one. CD8(+) T cell interference (following MVA coadministration as a mixture) may be caused partly by a lack of physiologic space for high-magnitude responses against multiple Ags, but is not caused by competition for presentation of Ag on MHC class I molecules, nor is it due to restricted T cell access to APCs presenting both Ags. Instead, enhanced killing of peptide-pulsed cells is observed in mice possessing pre-existing T cells against two Ags compared with just one, suggesting that priming against multiple Ags may in part reduce the potency of multiantigen MVA vectors to stimulate secondary CD8(+) T cell responses. These data have important implications for the development of a multistage or multicomponent viral vectored malaria vaccine for use in humans.     

Successful induction of CD8 T cell-dependent protection against malaria by sequential immunization with DNA and recombinant poxvirus of neonatal mice born to immune mothers

In some parts of Africa, 50% of deaths attributed to malaria occur in infants less than 8 mo. Thus, immunization against malaria may have to begin in the neonatal period, when neonates have maternally acquired Abs against malaria parasite proteins. Many malaria vaccines in development rely upon CD8 cells as immune effectors. Some studies indicate that neonates do not mount optimal CD8 cell responses. We report that BALB/c mice first immunized as neonates (7 days) with a Plasmodium yoelii circumsporozoite protein (PyCSP) DNA vaccine mixed with a plasmid expressing murine GM-CSF (DG) and boosted at 28 days with poxvirus expressing PyCSP were protected (93%) as well as mice immunized entirely as adults (70%). Protection was dependent on CD8 cells, and mice had excellent anti-PyCSP IFN-gamma and cytotoxic T lymphocyte responses. Mice born of mothers previously exposed to P. yoelii parasites or immunized with the vaccine were protected and had excellent T cell responses. These data support assessment of this immunization strategy in neonates/young infants in areas in which malaria exacts its greatest toll.     

Extended immunization intervals enhance the immunogenicity and protective efficacy of plasmid DNA vaccines

Effective vaccines against infectious diseases and biological warfare agents remain an urgent public health priority. Studies have characterized the differentiation of effector and memory T cells and identified a subset of T cells capable of conferring enhanced protective immunity against pathogen challenge. We hypothesized that the kinetics of T cell differentiation influences the immunogenicity and protective efficacy of plasmid DNA vaccines, and tested this hypothesis in the Plasmodium yoelii murine model of malaria. We found that increasing the interval between immunizations significantly enhanced the frequency and magnitude of CD8+ and CD4+ T cell responses as well as protective immunity against sporozoite challenge. Moreover, the interval between immunizations was more important than the total number of immunizations. Immunization interval had a significantly greater impact on T cell responses and protective immunity than on antibody responses. With prolonged immunization intervals, T cell responses induced by homologous DNA only regimens achieved levels similar to those induced by heterologous DNA prime/ virus boost immunization at standard intervals. Our studies establish that the dosing interval significantly impacts the immunogenicity and protective efficacy of plasmid DNA vaccines.     

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.     

Enhancing blood-stage malaria subunit vaccine immunogenicity in rhesus macaques by combining adenovirus, poxvirus, and protein-in-adjuvant vaccines

Protein-in-adjuvant formulations and viral-vectored vaccines encoding blood-stage malaria Ags have shown efficacy in rodent malaria models and in vitro assays against Plasmodium falciparum. Abs and CD4(+) T cell responses are associated with protective efficacy against blood-stage malaria, whereas CD8(+) T cells against some classical blood-stage Ags can also have a protective effect against liver-stage parasites. No subunit vaccine strategy alone has generated demonstrable high-level efficacy against blood-stage infection in clinical trials. The induction of high-level Ab responses, as well as potent T and B cell effector and memory populations, is likely to be essential to achieve immediate and sustained protective efficacy in humans. This study describes in detail the immunogenicity of vaccines against P. falciparum apical membrane Ag 1 in rhesus macaques (Macaca mulatta), including the chimpanzee adenovirus 63 (AdCh63), the poxvirus modified vaccinia virus Ankara (MVA), and protein vaccines formulated in Alhydrogel or CoVaccine HT adjuvants. AdCh63-MVA heterologous prime-boost immunization induces strong and long-lasting multifunctional CD8(+) and CD4(+) T cell responses that exhibit a central memory-like phenotype. Three-shot (AdCh63-MVA-protein) or two-shot (AdCh63-protein) regimens induce memory B cells and high-titer functional IgG responses that inhibit the growth of two divergent strains of P. falciparum in vitro. Prior immunization with adenoviral vectors of alternative human or simian serotype does not affect the immunogenicity of the AdCh63 apical membrane Ag 1 vaccine. These data encourage the further clinical development and coadministration of protein and viral vector vaccine platforms in an attempt to induce broad cellular and humoral immune responses against blood-stage malaria Ags in humans.     

Adjuvant-like effect of vaccinia virus 14K protein: a case study with malaria vaccine based on the circumsporozoite protein

Development of subunit vaccines for malaria that elicit a strong, long-term memory response is an intensive area of research, with the focus on improving the immunogenicity of a circumsporozoite (CS) protein-based vaccine. In this study, we found that a chimeric protein, formed by fusing vaccinia virus protein 14K (A27) to the CS of Plasmodium yoelii, induces strong effector memory CD8(+) T cell responses in addition to high-affinity Abs when used as a priming agent in the absence of any adjuvant, followed by an attenuated vaccinia virus boost expressing CS in murine models. Moreover, priming with the chimeric protein improved the magnitude and polyfunctionality of cytokine-secreting CD8(+) T cells. This fusion protein formed oligomers/aggregates that led to activation of STAT-1 and IFN regulatory factor-3 in human macrophages, indicating a type I IFN response, resulting in NO, IL-12, and IL-6 induction. Furthermore, this vaccination regimen inhibited the liver stage development of the parasite, resulting in sterile protection. In summary, we propose a novel approach in designing CS based pre-erythrocytic vaccines against Plasmodium using the adjuvant-like effect of the immunogenic vaccinia virus protein 14K.     

A candidate HIV/AIDS vaccine (MVA-B) lacking vaccinia virus gene C6L enhances memory HIV-1-specific T-cell responses

The vaccinia virus (VACV) C6 protein has sequence similarities with the poxvirus family Pox_A46, involved in regulation of host immune responses, but its role is unknown. Here, we have characterized the C6 protein and its effects in virus replication, innate immune sensing and immunogenicity in vivo. C6 is a 18.2 kDa protein, which is expressed early during virus infection and localizes to the cytoplasm of infected cells. Deletion of the C6L gene from the poxvirus vector MVA-B expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (MVA-B ΔC6L) had no effect on virus growth kinetics; therefore C6 protein is not essential for virus replication. The innate immune signals elicited by MVA-B ΔC6L in human macrophages and monocyte-derived dendritic cells (moDCs) are characterized by the up-regulation of the expression of IFN-β and IFN-α/β-inducible genes. In a DNA prime/MVA boost immunization protocol in mice, flow cytometry analysis revealed that MVA-B ΔC6L enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4+ and CD8+ T-cell memory immune responses, with most of the HIV-1 responses mediated by the CD8+ T-cell compartment with an effector phenotype. Significantly, while MVA-B induced preferentially Env- and Gag-specific CD8+ T-cell responses, MVA-B ΔC6L induced more Gag-Pol-Nef-specific CD8+ T-cell responses. Furthermore, MVA-B ΔC6L enhanced the levels of antibodies against Env in comparison with MVA-B. These findings revealed that C6 can be considered as an immunomodulator and that deleting C6L gene in MVA-B confers an immunological benefit by enhancing IFN-β-dependent responses and increasing the magnitude and quality of the T-cell memory immune responses to HIV-1 antigens. Our observations are relevant for the improvement of MVA vectors as HIV-1 vaccines.     

Splenic dendritic cell subsets prime and boost CD8 T cells and are involved in the generation of effector CD8 T cells

The ability of the dendritic cell (DC) subsets, CD8alpha+ and CD8alpha- DCs, to initiate a CD8 T cell response or to activate memory CD8 T cells and generate effector CD8 T cells has been controversial. In this study, we analyse the capacity of splenic DC subsets to induce CD8 T cell responses to a CD8 T cell epitope (pb9) of a malaria antigen. The administration of peptide-pulsed CD8alpha- or CD8alpha+ DCs primes and boosts a primed CD8 T cell response against the malaria epitope. In vitro, depletion of CD11c(+) DCs from mouse splenocytes, immunised with recombinant vaccinia virus Ankara (MVA) expressing pb9 epitope, significantly reduced the generation of pb9-specific IFNgamma producing effector CD8 T cells, indicating that splenic DCs are involved in the development of pb9-specific IFNgamma producing effector cells. Taken together, this result shows that both DC subsets have the ability to prime and boost CD8 T cell responses and are involved in the activation of memory CD8 T cells.     

CD8+ T lymphocytes protective against malaria liver stages are primed in skin-draining lymph nodes

The success of immunization with irradiated sporozoites is unparalleled among the current vaccination approaches against malaria, but its mechanistic underpinnings have yet to be fully elucidated. Using a model mimicking natural infection by Plasmodium yoelii, we delineated early events governing the development of protective CD8(+) T-cell responses to the circumsporozoite protein. We demonstrate that dendritic cells in cutaneous lymph nodes prime the first cohort of CD8(+) T cells after an infectious mosquito bite. Ablation of these lymphoid sites greatly impairs subsequent development of protective immunity. Activated CD8(+) T cells then travel to systemic sites, including the liver, in a sphingosine-1-phosphate (S1P)-dependent fashion. These effector cells, however, no longer require bone marrow-derived antigen-presenting cells for protection; instead, they recognize antigen on parenchymal cells-presumably parasitized hepatocytes. Therefore, we report an unexpected dichotomy in the tissue restriction of host responses during the development and execution of protective immunity to Plasmodium.     

Rapid memory CD8+ T-lymphocyte induction through priming with recombinant Mycobacterium smegmatis

The most promising vaccine strategies for the induction of cytotoxic-T-lymphocyte responses have been heterologous prime/boost regimens employing a plasmid DNA prime and a live recombinant-vector boost. The priming immunogen in these regimens must elicit antigen-specific memory CD8+ T lymphocytes that will expand following the boosting immunization. Because plasmid DNA immunogens are expensive and their immunogenicity has proven disappointing in human clinical trials, we have been exploring novel priming immunogens that might be used in heterologous immunization regimens. Here we show that priming with a prototype recombinant Mycobacterium smegmatis strain expressing human immunodeficiency virus type 1 (HIV-1) gp120-elicited CD4+ T lymphocytes with a functional profile of helper cells as well as a CD8+ T-lymphocyte population. These CD8+ T lymphocytes rapidly differentiated to memory cells, defined on the basis of their cytokine profile and expression of CD62L and CD27. Moreover, these recombinant-mycobacterium-induced T lymphocytes rapidly expanded following boosting with a recombinant adenovirus expressing HIV-1 Env to gp120-specific CD8+ T lymphocytes. This work demonstrates a remarkable skewing of recombinant-mycobacterium-induced T lymphocytes to durable antigen-specific memory CD8+ T cells and suggests that such immunogens might be used as priming vectors in prime/boost vaccination regimens for the induction of cellular immune responses.     

Novel protein and poxvirus-based vaccine combinations for simultaneous induction of humoral and cell-mediated immunity

The presence of both cell-mediated and humoral immunity is important in protection from and clearance of a number of infectious pathogens. We describe novel vaccine regimens using combinations of plasmid DNA, poxvirus and protein to induce strong Ag-specific T cell and Ab responses simultaneously in a murine model. Intramuscular (i.m.) immunization with plasmid DNA encoding the middle Ag of hepatitis B (DNA) concurrently with a commercial hepatitis B virus (HBV) vaccine (Engerix-B) followed by boosting immunizations with both modified vaccinia virus Ankara (MVA) encoding the middle Ag of HBV and Engerix-B induced high levels of CD4(+) and CD8(+) T cells and high titer Ab responses to hepatitis B surface Ag (HbsAg). Substitution of Engerix-B with adjuvant-free rHBsAg induced similar T cell responses and greatly enhanced Ab levels. Repeated immunizations with recombinant or nonrecombinant MVA mixed with Ag induced higher titers of Abs compared with immunization with either Ag or Engerix-B further demonstrating this novel adjuvant effect of MVA. The poxviruses NYVAC, fowlpox (FP9) and ALVAC, and to a lesser extent, adenovirus, also displayed similar adjuvant properties when used in combination with rHBsAg. The use of poxviruses as an adjuvant for protein to concurrently induce Ag-specific T cells and Abs could be applied to the development of vaccines for many diseases, including HIV and malaria, where both cell mediated and humoral immunity may be important for protection.     

DNA-based vaccines against malaria: status and promise of the Multi-Stage Malaria DNA Vaccine Operation

The introduction of DNA vaccine technology has facilitated an unprecedented multi-antigen approach to developing an effective vaccine against complex pathogens such as the Plasmodium spp. parasites that cause malaria. We have established the capacity of DNA vaccines encoding Plasmodium antigens to induce CD8(+) cytotoxic T lymphocyte and interferon-gamma responses in mice, monkeys and humans. However, like others, we have found that the first or second generation DNA vaccines on their own are not optimal, and have demonstrated the potential of heterologous prime/boost immunisation strategies involving priming with DNA and boosting with poxvirus or recombinant protein in adjuvant. In this review, we summarise the current status and promise of our programmatic efforts to develop a DNA-based vaccine against malaria, our Multi-Stage Malaria DNA Vaccine Operation, and illustrate the transition of promising developments in the laboratory to clinical assessment in humans.     

Deletion of Specific Immune-Modulatory Genes from Modified Vaccinia Virus Ankara-Based HIV Vaccines Engenders Improved Immunogenicity in Rhesus Macaques

Modified vaccinia virus Ankara (MVA) is a safe, attenuated orthopoxvirus that is being developed as a vaccine vector but has demonstrated limited immunogenicity in several early-phase clinical trials. Our objective was to rationally improve the immunogenicity of MVA-based HIV/AIDS vaccines via the targeted deletion of specific poxvirus immune-modulatory genes. Vaccines expressing codon-optimized HIV subtype C consensus Env and Gag antigens were generated from MVA vector backbones that (i) harbor simultaneous deletions of four viral immune-modulatory genes, encoding an interleukin-18 (IL-18) binding protein, an IL-1β receptor, a dominant negative Toll/IL-1 signaling adapter, and CC-chemokine binding protein (MVAΔ4-HIV); (ii) harbor a deletion of an additional (fifth) viral gene, encoding uracil-DNA glycosylase (MVAΔ5-HIV); or (iii) represent the parental MVA backbone as a control (MVA-HIV). We performed head-to-head comparisons of the cellular and humoral immune responses that were elicited by these vectors during homologous prime-boost immunization regimens utilizing either high-dose (2 × 10⁸ PFU) or low-dose (1 × 10⁷ PFU) intramuscular immunization of rhesus macaques. At all time points, a majority of the HIV-specific T cell responses, elicited by all vectors, were directed against Env, rather than Gag, determinants, as previously observed with other vector systems. Both modified vectors elicited up to 6-fold-higher frequencies of HIV-specific CD8 and CD4 T cell responses and up to 25-fold-higher titers of Env (gp120)-specific binding (nonneutralizing) antibody responses that were relatively transient in nature. While the correlates of protection against HIV infection remain incompletely defined, our results indicate that the rational deletion of specific genes from MVA vectors can positively alter their cellular and humoral immunogenicity profiles in nonhuman primates.     

Plasmodium-host interactions directly influence the threshold of memory CD8 T cells required for protective immunity

Plasmodium infections are responsible for millions of cases of malaria and ～1 million deaths annually. Recently, we showed that sterile protection (95%) in BALB/c mice required Plasmodium berghei circumsporozoite protein (CS(252-260))-specific memory CD8 T cells exceeding a threshold of 1% of all PBLs. Importantly, it is not known if Plasmodium species affect the threshold of CS-specific memory CD8 T cells required for protection. Furthermore, C57BL/6 mice immunized with radiation-attenuated parasites are more difficult to protect against Plasmodium sporozoite challenge than similarly immunized BALB/c mice; however, it is not known whether this is the result of different CD8 T cell specificity, functional attributes of CD8 T cells, or mouse strain-specific factors expressed in nonhematopoietic cells. In this article, we show that more CS-specific memory CD8 T cells are required for protection against P. yoelii sporozoite challenge than for protection against P. berghei sporozoite challenge. Furthermore, P. berghei CS(252)-specific CD8 T cells exhibit reduced protection against P. berghei sporozoite challenge in the context of C57BL/6 and C57BL/10 non-MHC-linked genes in CB6F1 and B10.D2 mice, respectively. Generation and immunization of reciprocal chimeric mice between BALB/c and B10.D2 strains revealed that B10 background factors expressed by nonhematopoietic cells increased the threshold required for protection through a CD8 T cell-extrinsic mechanism. Finally, reduced CS-specific memory CD8 T cell protection in P. yoelii-infected BALB/c or P. berghei-infected B10.D2 mice correlated with increased rates of Plasmodium amplification in the liver. Thus, both Plasmodium species and strain-specific background genes in nonhematopoietic cells determine the threshold of memory CD8 T cells required for protection.     

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.     

Kunjin virus replicon vectors for human immunodeficiency virus vaccine development

We have previously demonstrated the ability of the vaccine vectors based on replicon RNA of the Australian flavivirus Kunjin (KUN) to induce protective antiviral and anticancer CD8+ T-cell responses using murine polyepitope as a model immunogen (I. Anraku, T. J. Harvey, R. Linedale, J. Gardner, D. Harrich, A. Suhrbier, and A. A. Khromykh, J. Virol. 76:3791-3799, 2002). Here we showed that immunization of BALB/c mice with KUN replicons encoding HIV-1 Gag antigen resulted in induction of both Gag-specific antibody and protective Gag-specific CD8+ T-cell responses. Two immunizations with KUNgag replicons in the form of virus-like particles (VLPs) induced anti-Gag antibodies with titers of > or =1:10,000. Immunization with KUNgag replicons delivered as plasmid DNA, naked RNA, or VLPs induced potent Gag-specific CD8+ T-cell responses, with one immunization of KUNgag VLPs inducing 4.5-fold-more CD8+ T cells than the number induced after immunization with recombinant vaccinia virus carrying the gag gene (rVVgag). Two immunizations with KUNgag VLPs also provided significant protection against challenge with rVVgag. Importantly, KUN replicon VLP vaccinations induced long-lasting immune responses with CD8+ T cells able to secrete gamma interferon and to mediate protection 6 to 10 months after immunization. These results illustrate the potential value of the KUN replicon vectors for human immunodeficiency virus vaccine design.     

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.     

Heterologous prime/boost immunization of rhesus monkeys by using diverse poxvirus vectors

As the diversity of potential immunogens increases within certain classes of vectors, the possibility has arisen of employing heterologous prime/boost immunizations using diverse members of the same family of vectors. The present study was initiated to explore the use of divergent pox vectors in a prime/boost regimen to elicit high-frequency cellular immune responses to human immunodeficiency virus type 1 envelope and simian immunodeficiency virus gag in rhesus monkeys. We demonstrated that monkeys vaccinated with a recombinant modified vaccinia virus Ankara (rMVA) prime/recombinant fowlpox virus (rFPV) boost regimen and monkeys vaccinated with a recombinant vaccinia virus prime/rFPV boost regimen developed comparable cellular immune responses that were greater in magnitude than those elicited by a homologous prime/boost with rMVA. Nevertheless, comparable magnitude recall cellular immune responses were observed in monkeys vaccinated with heterologous and homologous recombinant poxvirus following challenge with the CXCR4-tropic SHIV-89.6P. Consistent with this finding, comparable levels of containment of viral replication and CD4(+) T-lymphocyte preservation were seen in these groups of recombinant poxvirus-vaccinated monkeys. This study supports further exploration of combining recombinant vectors of the same family in prime/boost immunization strategies to optimize vaccine-elicited cellular immune responses.