Humanized mice reconstituted with human hematopoietic cells have been developed as an experimental animal model for human immunodeficiency virus type 1 (HIV-1) infection. Myeloablative irradiation is usually performed to augment the engraftment of donor hematopoietic stem cells (HSCs) in recipient mice; however, some mouse strains are susceptible to irradiation, making longitudinal analysis difficult. We previously attempted to construct humanized NOD/SCID/JAK3null (hNOJ) mice, which were not irradiated prior to human HSC transplantation. We found that, over time, many of the reconstituted CD4+ T cells expanded with an activated effector memory phenotype. Therefore, the present study used hNOJ mice that were irradiated (hNOJ (IR+)) or not (hNOJ (IR−)) prior to human HSC transplantation to examine whether the development and cellularity of the reconstituted CD4+ T cells were influenced by the degree of chimerism, and whether they affected HIV-1 infectivity. Indeed, hNOJ (IR+) mice showed a greater degree of chimerism than hNOJ (IR−) mice. However, the conversion of CD4+ T cells to an activated effector memory phenotype, with a high percentage of cells showing Ki-67 expression, occurred in both hNOJ (IR+) and hNOJ (IR−) mice, probably as a result of lymphopenia-induced homeostatic expansion. Furthermore, when hNOJ (IR+) and hNOJ (IR−) mice, which were selected as naïve- and memory CD4+ T cell subset-rich groups, respectively, were infected with CCR5-tropic HIV-1 in vivo, virus replication (as assessed by the plasma viral load) was delayed; however, the titer subsequently reached a 1-log higher level in memory-rich hNOJ (IR−) mice than in naïve-rich hNOJ (IR+) mice, indicating that virus infectivity in hNOJ mice was affected by the different status of the reconstituted CD4+ T cells. Therefore, the hNOJ mouse model should be used selectively, i.e., according to the specific experimental objectives, to gain an appropriate understanding of HIV-1 infection/pathogenesis.

Flow cytometric analysis is a reliable and convenient method for investigating molecules at the single cell level. Previously, recombinant human immunodeficiency virus type 1 (HIV-1) strains were constructed that express a fluorescent reporter, either enhanced green fluorescent protein, or DsRed, which allow the monitoring of HIV-1-infected cells by flow cytometry. The present study further investigated the potential of these recombinant viruses in terms of whether the HIV-1 fluorescent reporters would be helpful in evaluating viral replication based on fluorescence intensity. When primary CD4+ T cells were infected with recombinant viruses, the fluorescent reporter intensity measured by flow cytometry was associated with the level of CD4 downmodulation and Gag p24 expression in infected cells. Interestingly, some HIV-1-infected cells, in which CD4 was only moderately downmodulated, were reporter-positive but Gag p24-negative. Furthermore, when the activation status of primary CD4+ T cells was modulated by T cell receptor-mediated stimulation, we confirmed the preferential viral production upon strong stimulation and showed that the intensity of the fluorescent reporter within a proportion of HIV-1-infected cells was correlated with the viral replication level. These findings indicate that a fluorescent reporter encoded within HIV-1 is useful for the sensitive detection of productively infected cells at different stages of infection and for evaluating cell-associated viral replication at the single cell level.

Oral immunization is a safe and easily applicable route to induce mucosal immunity to HIV infection. We examined the ability of oral attenuated Salmonella typhimurium (ST) vaccine expressing Gag for the efficiency of generating Gag-specific mucosal IgA and CD8+ T cell responses in intestinal lymphoid tissues. By optimizing the codon of HIV-1 gag to the preferred codon bias of Salmonella, the expression of Gag in Salmonella was dramatically improved. The oral ST-Gag vaccine by itself was not so powerful and induces little Gag-specific CD8+ T cell responses in the intestine. Nevertheless, we found that it potentiates otherwise weak intestinal CD8+ T cell responses in nasally primed mice with Gag p24 and cholera toxin adjuvant. Thus, the oral delivery of Salmonella expressing Gag would be utilized in combination with other parenteral vaccine to direct and strengthen intestinal HIV-specific CTL responses.