Dendritic cells are less susceptible to human immunodeficiency virus type 2 (HIV-2) infection than to HIV-1 infection.

Human immunodeficiency virus type 1 (HIV-1) infection of dendritic cells (DCs) has been documented in vivo and may be an important contributor to HIV-1 transmission and pathogenesis. HIV-1-specific CD4(+) T cells respond to HIV antigens presented by HIV-1-infected DCs and in this process become infected, thereby providing a mechanism through which HIV-1-specific CD4(+) T cells could become preferentially infected in vivo. HIV-2 disease is attenuated with respect to HIV-1 disease, and host immune responses are thought to be contributory. Here we investigated the susceptibility of primary myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to infection by HIV-2. We found that neither CCR5-tropic primary HIV-2 isolates nor a lab-adapted CXCR4-tropic HIV-2 strain could efficiently infect mDCs or pDCs, though these viruses could infect primary CD4(+) T cells in vitro. HIV-2-exposed mDCs were also incapable of transferring virus to autologous CD4(+) T cells. Despite this, we found that HIV-2-specific CD4(+) T cells contained more viral DNA than memory CD4(+) T cells of other specificities in vivo. These data suggest that either infection of DCs is not an important contributor to infection of HIV-2-specific CD4(+) T cells in vivo or that infection of DCs by HIV-2 occurs at a level that is undetectable in vitro. The frequent carriage of HIV-2 DNA within HIV-2-specific CD4(+) T cells, however, does not appear to be incompatible with preserved numbers and functionality of HIV-2-specific CD4(+) T cells in vivo, suggesting that additional mechanisms contribute to maintenance of HIV-2-specific CD4(+) T-cell help in vivo.

Infectivity of herpes simplex virus type-1 (HSV-1) amplicon vectors in dendritic cells is determined by the helper virus strain used for packaging.

Herpes simplex virus type-1 (HSV-1) amplicon vectors are being explored for a wide range of potential applications, including vaccine delivery and immunotherapy of cancer. While extensive effort has been directed towards the improvement of the amplicon "payload" in these vectors, relatively little attention has been paid to the effect of the packaging HSV-1 strains on the biological properties of co-packaged amplicon vectors. We therefore compared the biological properties of amplicon stocks prepared using a panel of primary HSV-1 isolates, a molecularly cloned strain used to package helper-free amplicons (designated here as F5), and two laboratory isolates (KOS and strain 17, which is the parent of the F5 clone). This analysis revealed considerable inter-strain variability in the ability of amplicon stocks packaged by different primary HSV-1 isolates to efficiently transduce established cell lines and primary human dendritic cells (DC). Amplicons packaged by both the F5 molecularly cloned virus and its laboratory-adapted parent (strain 17) were very inefficient at transducing DC, when compared to amplicons packaged by KOS or by several of the primary virus isolates. These finding have important implications for the future development of improved amplicon-based vaccine delivery systems and suggest that DC tropism may be an instrinsic property of some HSV-1 strains, independent of passage history or molecular cloning.

Spatial and temporal expression of herpes simplex virus type 1 amplicon-encoded genes: implications for their use as immunization vectors.

There is great interest in developing new immunization vectors. Helper virus-free herpes amplicons, plasmid-based vectors that encode no viral gene products and have an extremely large coding capacity, are attractive viral vaccine candidates for expressing recombinant proteins in vivo for immunization. Earlier studies in mice, using amplicons encoding the gp120 protein of human immunodeficiency virus (HIV), resulted in strikingly robust cellular immune responses as measured by cytotoxicity and interferon gamma enzyme-linked immunospot assays. To begin to understand how such vectors function in vivo to generate an immune response, we used amplicons encoding reporter constructs including green fluorescent protein (GFP) and luciferase to examine the duration of expression after administration to mice. Luciferase expression, measured with the IVIS system from Xenogen/Caliper Life Sciences (Hopkinton, MA) and by enzymatic assays of tissue extracts, revealed that expression after injection of the HSVluc amplicons peaked earlier than 24 hr after injection into mice. HSVegfp injection resulted in peak accumulation of GFP 24 hr after administration in vivo. Thus, both reporter genes revealed a rather rapid and robust expression pattern of short duration. The short period of expression appears in part to be due to gene silencing. Examination of the cells transduced by amplicons encoding GFP and human B7.1 suggested that the amplicons transduce a variety of cells, including professional antigen-presenting cells. From this and previous work, we conclude that amplicons may engender a potent immune response by directly transducing dendritic cells as well as by cross-priming of antigen produced by other transduced host cells.

Effect of promoter strength on protein expression and immunogenicity of an HSV-1 amplicon vector encoding HIV-1 Gag.

Helper-free herpes simplex virus type-1 (HSV-1) amplicon vectors elicit robust immune responses to encoded proteins, including human immunodeficiency virus type-1 (HIV-1) antigens. To improve this vaccine delivery system, seven amplicon vectors were constructed, each encoding HIV-1 Gag under the control of a different promoter. Gag expression levels were analyzed in murine and human cell lines, as well as in biopsied tissue samples from injected mice; these data were then compared with Gag-specific T cell responses in BALB/c mice. The magnitude of the amplicon-induced immune response was found to correlate strongly with the level of Gag production both in vitro and in vivo. Interestingly, the best correlation of the strength of the amplicon-induced immune response was with antigen expression in cultured DC rather than expression at the tissue site of injection or in cultured cell lines. These findings may have implications for the generation of improved HSV-1 amplicon vectors for HIV-1 vaccine delivery.

Amplicons as vaccine vectors.

HSV-1 amplicon vectors efficiently transduce cultured antigen-presenting cells (APC), including both human and murine dendritic cells as well as primary human chronic lymphocytic leukemia (CLL) B cells. Helper-free amplicons have been shown to be especially well-suited for this purpose, since they do not impair the antigen-presenting functions of these target cells. In vivo, amplicon vectors have been used in preclinical studies aimed at the development of therapeutic cancer vaccines, as well as vaccines for Alzheimer's disease, and selected microbial pathogens. Studies in small animal model systems have shown that ex vivo transduction of irradiated tumor cells with amplicon vectors encoding immunomodulatory cytokines such as IL-2 or GM-CSF can elicit protective responses against a tumor challenge. In an experimental model for cancer immunotherapy, direct transduction of preformed tumors with vectors encoding CD40L resulted in slowed tumor growth or tumor eradication. Other studies have examined the ability of amplicons to elicit immune responses against encoded antigens, and have shown that strong cellular immune responses can be generated against amplicon encoded HIV-1 antigens in mice. Thus, amplicon vectors have shown significant promise as vaccine vectors in a range of settings. These promising initial findings highlight the need to perform additional studies, including experiments to evaluate the immunogenicity of amplicon vectors in additional animal models, possibly including nonhuman primates. Overall, amplicon vectors offer compelling advantages when compared to other vaccine-delivery platforms, which include the capacity to incorporate a very large transgene payload and the potential to efficiently transduce mucosal surfaces. It will be important to design future studies to directly test and exploit these features of the amplicon system. The next few years therefore promise to be an exciting and important period in the development of amplicons as vaccine vectors.

Human dendritic cells transduced with herpes simplex virus amplicons encoding human immunodeficiency virus type 1 (HIV-1) gp120 elicit adaptive immune responses from human cells engrafted into NOD/SCID mice and confer partial protection against HIV-1 challenge.

Small-animal models are needed to test human immunodeficiency virus (HIV) vaccine efficacy following viral challenge. To this end, we examined HIV-1-specific immune responses following immunization of nonobese diabetic-severe combined immunodeficient mice that were repopulated with human peripheral blood lymphocytes (hu-PBL-NOD/SCID mice). Autologous dendritic cells (DC) were transduced ex vivo with replication-defective, helper virus-free, herpes simplex virus type 1 (HSV-1) amplicons that expressed HIV-1 gp120 and were then injected into the hu-PBL-NOD/SCID mice. This resulted in primary HIV-1-specific humoral and cellular immune responses. Serum samples from vaccinated animals contained human immunoglobulin G that reacted with HIV-1 Env proteins by enzyme-linked immunosorbent assay and neutralized the infectivity of HIV-1 LAI and ADA strains. T cells isolated from the mice responded to viral antigens by producing gamma interferon when analyzed by enzyme-linked immunospot assay. Importantly, exposure of the vaccinated animals to infectious HIV-1 demonstrated partial protection against infectious HIV-1 challenge. This was reflected by a reduction in HIV-1(ADA) and by protection of the engrafted human CD4(+) T lymphocytes against HIV-1(LAI)-induced cytotoxicity. These data demonstrate that transduction of DC by HSV amplicon vectors expressing HIV-1 gp120 induce virus-specific immune responses in hu-PBL-NOD/SCID mice. This mouse model may be a useful tool to evaluate human immune responses and protection against viral infection following vaccination.

Murine cytomegalovirus abortively infects human dendritic cells, leading to expression and presentation of virally vectored genes.

Dendritic cells (DC) are potent antigen-presenting cells that play a crucial role in antigen-specific immune responses. Thus, the targeting of exogenous antigens to DC has become a popular approach for cancer immunotherapy and vaccine development. In this report, we studied the interplay between murine cytomegalovirus (MCMV) and human monocyte-derived DC. The results showed that an enhanced green fluorescent protein (EGFP)-encoding, replication-competent MCMV vector underwent abortive infection in human DC; this was accompanied by the efficient expression of EGFP. Infection of human DC by this vector resulted in a modest increase in the expression of cell surface proteins associated with DC maturation and has no significant effect on the immunostimulatory function of the cells, as reflected by their ability to support T-cell proliferation in a mixed-lymphocyte reaction. Finally, an MCMV vector encoding the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein was constructed and used to infect cultured human DC. The infected DC were shown to be capable of stimulating the expansion of autologous, gp120-specific, class I-restricted T lymphocytes from an HIV-1-negative donor, as determined by tetramer staining and enzyme-linked immunospot analysis. Taken together, these results suggest that MCMV may have potential utility as a vector for human vaccine development.