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. Author manuscript; available in PMC: 2021 Feb 23.
Published in final edited form as: Antiviral Res. 2017 Apr 7;144:83–92. doi: 10.1016/j.antiviral.2017.03.027

Early phase dynamics of traceable mCherry fluorescent protein-carrying HIV-1 infection in human peripheral blood mononuclear cells-transplanted NOD/SCID/Jak3−/− mice

Nobuyo Higashi-Kuwata a,b, Hiromi Ogata-Aoki b,c, Shin-ichiro Hattori a,d, Hironori Hayashi a,b, Matthew Danish b, Manabu Aoki b,c,e, Chiemi Shiotsu f, Tatsuyoshi Kawamura g, Hironobu Ihn f, Hisataka Kobayashi h, Seiji Okada d, Hiroaki Mitsuya a,b,c,*
PMCID: PMC7900919  NIHMSID: NIHMS1665598  PMID: 28392419

Abstract

We attempted to elucidate early-phase dynamics of HIV-1 infection using replication-competent, red-fluorescent-protein (mCherry)-labeled HIV-1JR-FL (HIVJR-FL mC) and NOD/SCID/Jak3−/− mice transplanted with Individual-A's human peripheral blood mononuclear cells (hPBMC)(hNOJ mice). On day 7 following HIVJR-FL mC inoculation, mCherry-signal-emitting infection foci were readily identified in the subserosa of 10 of 10 HIVJR-FL mC-inoculated hNOJ mice, although infection foci were not located without the mCherry signal in unlabeled HIV-1JR-FL-inoculated mice (n = 6). Even on day 14, infection foci were hardly located in the unlabeled HIV-1JR-FL-inoculated mice, while in all of 7 HIVJR-FL mC-inoculated hNOJ mice examined, mCherry-signal-emitting lymph nodes were easily identified, in which active viral replication was present. On day 14, a significantly larger number of mesenteric lymph nodes were seen in HIVJR-FL mC-exposed hNOJ mice than in HIVJR-FL mC-unexposed mice (P = 0.0025). The weights of mesenteric lymph nodes of those HIVJR-FL mC -exposed hNOJ mice were also greater than those of HIVJR-FL mC-unexposed mice (P = 0.0005). When hNOJ mice were inoculated with HIVJR-FL mC-exposed hPBMC from Individual-B, significantly greater viremia was seen than in cell-free HIVJR-FLmC-inoculated hNOJ mice as examined on day 7. In the lymph nodes of those mice inoculated with HIVJR-FLmC-exposed hPBMC from Individual-B, a substantial number of Individual-B's HIVJR-FLmC-infected cells were identified together with Individual-A's cells as examined on day 14. The present HIVJR-FL mC-infected mouse model represents the first system reported using traceable HIVJR-FLmC and human target cells, not using SIV or simian cells, which should be of utility in studies of early-phases of HIV-1 transmission and in evaluating the effects of potential agents for post-exposure and pre-exposure and pre-exposure prophylaxis.

Keywords: HIV propagation, In vivo imaging, Early phase dynamics, Cell-associated HIV infection

1. Introduction

Certain early phase events in lentivirus infection have been examined mostly using SIV, including the innovative early works (Pope and Haase, 2003; Haase, 2010; Santangelo et al., 2015). However, the nature of SIV is not exactly the same as that of HIV-1 (Zhu et al., 2001; Riddick et al., 2016; Asmal et al., 2016) and the detailed dynamics and spreading profiles of HIV-1 at early phases of initial infection remain to be determined. Experimental nonhuman primate models that make it possible to examine the effects of pre-and postexposure prophylaxis (PrEP and PEP, respectively) with antiretroviral agents have been reported. However, there is a problematic limitation that because of the sizes of nonhuman primate models weighting as much as a few to 10 kg, relatively large doses of test agents, which are generally costly especially in the early phases of development, are required (Tsai et al.,1995, 1998; Irvine et al., 2015; Stoddart et al., 2015). Various rodent models for HIV-1 infection and its treatment have been reported; however, determination and evaluation of the presence of HIV-1 in a variety of tissues and organs in such small animals are time-consuming and labor-intensive since the sites of HIV-1 replication are difficult to determine, thus enforcing to sacrifice all animals and to examine all the tissues and organs following the sacrifice (Sun et al., 2007; Denton et al., 2010; Nischang et al., 2012). In this regard, we have used fluorescent imagining techniques by employing the intermolecular fluorescent resonance energy transfer (FRET)-based HIV expression assay using HIV-1 clones with cyan or yellow fluorescent proteins and have demonstrated the inhibition of HIV-1 protease dimerization by HIV-1 protease inhibitor, darunavir (DRV) (Koh et al., 2007) and tipranavir (Aoki et al., 2012).

In the research area of HIV-1 infection and AIDS, the construction of humanized animal models through transplantation of human tissues or immunocompetent cells into severely immunocompromised (SCID) mice has allowed the development of small human tissue/cell-reconstituted animal systems (Van Duyne et al., 2009). In our previous study, we utilized humanized mice, non-obese diabetic (NOD)-SCID, interleukin 2 receptor gamma (IL-2Rgamma)-chain-knocked-out (NOG) mice, and established a human peripheral blood mononuclear cells (hPBMC)-transplanted R5 HIV-1JR-FL-infected NOG mouse and showed that the NOG mouse system serves as a small-animal AIDS model and that AK602, an experimental CCR5 inhibitor, blocks the infection and propagation of R5-HIV-1 in that mouse model (Nakata et al., 2005). In 2008, a group led by Okada developed human CD34+ cells-transplanted, NOD/SCID/Janus kinase 3 (Jak3) knockout (NOJ) mice (Okada et al., 2008). In 2009, using a further refined NOJ mouse model, in which massive and systemic HIV-1 infection occurs, human CD4+/CD8+ cell ratios significantly decrease, and high levels of HIV-1 viremia are achieved (Hattori et al., 2009), we demonstrated that a novel HIV-1 reverse transcriptase inhibitor, 4’-ethynyl-2-fluoro-2’-deoxyadenosine or EFdA, exerts potent anti-HIV-1 activity (Hattori et al., 2009; Nakata et al., 2007). EFdA has presently been in clinical trials as a promising therapeutic for HIV-1 infection and AIDS in the United States (Grobler et al., 2016).

In the present study, we attempted to establish a system that enables us to examine the dynamics of HIV-1 infection using currently available in vivo imaging technologies (Kosaka et al., 2009). Regarding a fluorescent protein reporter, we chose a red version of DsRed, mCherry, which matures more rapidly (t0.5 = 15 min) and completely with monomeric structure, has greater tolerance of N- and C-terminal fusions, has better transmission through tissues (Miyawaki et al., 2003). Thus, we employed mCherry as an imaging reporter of infectious HIV-1 and also employed the NOJ mouse system.

2. Materials and methods

2.1. Generation of a recombinant HIV-1 clone containing traceable fluorescence protein (FP), mCherry fluorescent protein

In the generation of a full-length molecular infectious HIV-1 clone containing mCherry fluorescent protein, the PCR-mediated recombination method was used as previously described (Fang et al.,1999). More details are shown in Supplemental Material. We refer to this infectious recombinant HIV-1 as HIVJR-FL mC (Fig. 1A). The expression of mCherry, HIV-1 p24, and Nef proteins of HIVJR-FL mC was examined with Western blotting as described in Supplemental Material.

Fig. 1. Generation of a recombinant HIV-1 clone containing mCherry florescent protein (HIVJR-FL mC).

Fig. 1.

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(A) Genetic map of HIV-1 containing the mCherry fluorescent protein-encoding gene. HIV-1JR-FL was constructed to contain mCherry fluorescent protein between gp41 and nef proteins. (B) Comparison of the replicative capacity between HIVJR-FL mc and HIV-1JR-FL in monocyte-derived macrophages/dendritic cells (Mϕ/DC). Monocytes were stimulated with rhGM-CSF (i) or rhM-CSF (ii). Note that HIVJR-FLmC propagated in the presence of either of the cytokines, although HIVJR-FL mC was slower in its propagation than HIV-1JR-FL. Data represent the mean values determined in triplicate. (C) The mCherry signal was detected as early as on day 3 following the exposure to HIVJR-FL mC (a: left panel) in Mϕ/DC, but not when the cells were exposed to HIV-1JR-FL (b: left panel) as examined under confocal laser microscopy. Stronger mCherry signals were observed when examined on day 11 (c: left panel) in HIVJR-FL mC-exposed Mϕ/DC, whereas no mCherry signal was detected in HIV-1JR-FL-infected Mϕ/DC (d: left panel). The general morphology of the cells was captured under the differential interference contrast microscopy (a-d: right panels). Scale bar denotes 10 μm. (D) The expression of mCherry, HIV-1 p24, and Nef proteins of HIVJR-FL mC. After 6 days following pHIVJR-FL mc transfection of COS-7 cells, the expression of mCherry/DsRed and HIV-1 p24 proteins was detected as assessed with western blot using the COS-7 cell lysates (a). Neither mCherry nor Nef protein was detected in HIVJR-FLmC virion lysates (b). (E) The mCherry-encoding gene persisted within PBMCs obatined from the HIVJR-FLmC-infected hNOJ mice until day 14 following HIVJR-FLmC inoculation, although deletion of the mCherry-encoding gene apparently occurred by day 21 after the inoculation. Lanes 1 to 4 show the PCR-amplified DNA products containing the mCherry-coding gene within the provirus in huPBMC.

2.2. Preparation of primary human monocyte-derived macrophages

Heparinized blood was collected from a healthy HLA-A2+/A31− donor (Individual-A) after an informed consent was obtained based on the institutional guidelines approved by Kumamoto University Graduate School of Life Sciences in accordance with the Declaration of Helsinki. The approval for this study was obtained from the Ethics Committee for Epidemiological and General Research at the Faculty of Life Science, Kumamoto University (Approval ID; 348). Monocyte-derived macrophages were prepared as previously described (Chihara et al., 2010, 2012). More details are described in Supplemental Material.

2.3. Transplantation of human PBMC into NOJ mice

NOD-SCID-Jak3−/− (NOJ) mice were established and maintained in the Center for Animal Resources and Development, Kumamoto University (Okada et al., 2008). The mice used in the present study were 16–20 weeks old at the time of transplantation of human PBMC isolated from an HIV-1-seronegative HLA-A2+/A31− donor (Individual-A). Individual A's PBMC-transplanted NOJ mice (hNOJ mice) were generated by methods previously described (Hattori et al., 2009). More details are described in Supplemental Material.

2.4. Inoculation of HIVJR-FL mC into individual A's PBMCtransplanted NOJ mice (hNOJ mice)

The transfection-derived virions (HIVJR-FL mC; 300 ng p24) were intraperitoneally inoculated into each mouse in 10 days after PBMC transplantation. On days 7 and 14 after HIVJR-FL mC inoculation, we performed in vivo imaging, immunohistological staining, and measurement of plasma p24 levels. More details are described in Supplemental Material.

2.5. Visualization and determination of HIVJR-FL mC infection in hNOJ mice and detection of HIV-1 p24 antigen in the plasma

The spectral fluorescence images were captured using Maestro™ and MVX10-Nuance™ in vivo imaging systems (PerkinElmer), eliminating auto-fluorescence and providing multicolor flexibility and accuracy for both visible and near-infrared labels using multispectral acquisition and analysis. More details are described in Supplemental Material.

2.6. Quantitative examination of lymph nodes

To determine the location and anatomical terminology of lymph nodes, we referred to “Patterns of lymphatic drainage in the adult laboratory rat” published by Tilney, (1971). More details are described in Supplemental Material.

2.7. Statistical analyses

Nonparametric statistical analyses were performed by using the Mann-Whitney U test (Statview, version 5.0; Abacus Concepts). Assessment of correlations was carried out using the Fisher Exact test (JMP® software; SAS Institute). P values of <0.05 were defined as significant.

3. Results

3.1. HIVJR-FL mC replicates and expresses mCherry protein in vitro and the mCherry-encoding gene persists within the provirus in HIVJR-FL mC-infected hPBMC until day 14 following HIVJR-FL mC inoculation of hNOJ mice

We first examined the replicative capacity of HIVJR-FL mC (Fig. 1A) in human monocyte-derived macrophages/dendritic cells (Mϕ/DC). In the presence of rhGM-CSF (10 μg/ml), HIV-1JR-FL replicated well without any other stimulations and HIVJR-FL mC also replicated albeit less vigorously under the same conditions (Fig.1Bi). In the presence of rhM-CSF (100 μg/ml), both HIV-1JR-FL and HIVJR-FL mC propagated relatively well, but the replication levels were less than those with rhGM-CSF (Fig. 1Bii). When Mϕ/DC were exposed to HIVJR-FL mC, the mCherry signal was detected in the target Mϕ/DC in as early as day 3 (Fig. 1Ca), and the majority of the Mϕ/DC had been positive for the mCherry signal by day 11 following the exposure (Fig. 1Cc). The expression of mCherry/DsRed and HIV-1 p24 proteins was detected as assessed with western blot of the COS-7 cell lysates (Fig. 1Da). While neither mCherry nor Nef protein was identified in the HIVJR-FL mc virion lysates (Fig. 1Db), HIVJR-FLmC continued replication and expressed mCherry protein within the HIVJR-FL mC-infected cells (Fig. 1Da), indicating that HIVJR-FL mC-infected cells and/or infection foci were to be located and traced. In fact, we confirmed that the mCherry-encoding gene was persistently identified within PBMC obatined from the HIVJR-FL mC-infected hNOJ mice until day 14 following HIVJR-FL mC inoculation (Fig. 1E), although deletion of the mCherry-encoding gene apparently occurred by day 21 after the inoculation (Fig. 1E). Using HIVJR-FL mC, we thus chose a 14-day duration in further in vivo experiments.

3.2. hPBMC widely populated in the immune system organs of hNOJ mice

In order to ask whether the transplanted hPBMC (isolated from Individual-A) populated in various organs of hNOJ mice, eleven different organs (brain, lung, liver, spleen, kidney, skin, muscle, intestine, testis, vagina, and lymph nodes) were immunohistologically examined (Fig. 2). More details are described in Supplemental Material.

Fig. 2. Reconstitution of hPBMC in organs of hNOJ mice.

Fig. 2.

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(A) Fixed/paraffin-embedded tissue sections were immunohistologically stained with anti-human CD45 antibody (a pan-lymphocyte marker) and examined under light microscopy. Nuclei were counterstained with Mayer's hematoxylin (blue). Note that human CD45+ cells (brown) were abundantly seen in each of the organs examined including brain (a), lungs (b), liver (c), spleen (d), kidneys (e), skin (f), muscle (g), intestines (h), testis (i), vagina (j), and lymph nodes (k). Note that murine lymphocytes are virtually absent, human lymphocytes (hCD45+) are sparse, and no follicles were seen in the lymph nodes excised from the hNOJ mouse (k), while murine lymphocytes (hCD45−) are abundantly seen in lymph nodes excised from a healthy BALB/c mouse (l). Scale bars in (h) through (j) denote 200 μm and all others 20 μm. (B) Fixed/paraffin-embedded tissue sections were stained with anti-human CD20 (hCD20) antibody (a B lymphocyte marker). Note that hCD20+ cells (brown) were seen only in spleen (d) and lymph nodes (k) as indicated with arrows. Panel (l) shows the lymph node histology of a healthy BALB/c mouse, where no hCD20+ cells were seen. Scale bars in (h) through (j) denote 200 mm and all others 20 μm.

3.3. Visualization of HIVJR-FL mC in the hNOJ mice exposed to HIVJRFL mC

When hNOJ mice were inoculated with HIVJR-FL mC (Fig. 3), by day 7 following the exposure, a relatively dim mCherry signal was detected in the abdomen of all of the mice studied (n = 10) as examined with wavelength-resolved spectral imaging system (Maestro™). Three of the 10 mice were sacrificed on day 7 following the exposure and mCherry-positive organs were excised out. Examination of the excised organs under wavelength-resolved spectral imaging high-resolution stereomicroscopy (MVX10-Nuance™) revealed that the mCherry signal observed with Maestro™ was located in the serous membrane of the omentum (Fig. 4Aa-Ab). When p24 amounts in the plasma of the hNOJ mice (n = 10) were determined on day 7 following HIVJR-FL mC inoculation, no p24 was detected in 7 of the 10 mice, while moderate levels of p24 (~103 pg/ml) were detected in 3 mice (Fig. 4B). On day 14 following the exposure, two stronger and more concentrated mCherry signals were detected in the abdomen of the hNOJ mice with Maestro™ (n = 7). In the excised abdominal organs of the 14-day-sacrificed mice, the mCherry signal was located mostly in lymphoid organs as assessed with MVX10-Nuance™ (Fig. 4Ac-Ad). On day 14, the plasma samples from the remaining 7 mice contained significantly higher levels of p24 (~103-106 pg/ml) as compared to those on day 7 (P = 0.0012), indicating that the HIVJR-FL mC continuously replicated in the mice (Fig. 4B). When the 7-day dimly mCherry-positive serous membrane of the omentum was fixed with paraformaldehyde and subjected to immunohistological examination, both mCherry- and p24-poisitive cells were detected in the subserous layer of the omentum (Fig. 4Ca-Cb). As expected, greater numbers of mCherry-and p24-positive cells were identified in the lymphoid organs excised on day 14 than in those on day 7 (Fig. 4Ce-Cf). These cells also proved to be positive for both CD4 and CD68 (Fig. 4Da-Dc), indicating that the cells were mostly of the Mϕ/DC lineage. A few CD8 cells were also observed in some lymph nodes, but they were negative for p24 (Fig. 4Dd).

Fig. 3.

Fig. 3.

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Protocol for radiation, hPBMC transplantation, HIVJR-FL mC inoculation, and monitoring the dynamics of HIVJR-FL mC infection.

Fig. 4. Visualization of expanding HIVJR-FL mC infection in the HIVJR-FL mC-exposed hNOJ mice and detection of HIV-1 p24 antigen in the plasma.

Fig. 4.

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(A) mCherry fluorescent protein signals were seen only in the omentum area in all HIVJR-FL mC-exposed hNOJ mice examined on day 7 after HIVJR-FL mC inoculation and no lymph nodes were identified. However, on day 14 after the exposure, enlarged lymph nodes were seen with distinct mCherry fluorescent signals. Panels a and c show mCherry signals alone (at wave lengths 587–610 nm) and panels b and d show mCherry signals superimposed with light microscopy images. Arrows indicate the mCherry signals. Scale bars denote 1 cm. (B) HIV-1 p24 antigen in plasma from HIVJR-FL mC-exposed hNOJ mice. Plasma samples from the HIVJR-FL mC-infected mice were analyzed by the fully automated chemiluminescent enzyme immunoassay system on days 7 (n = 10) and 14 (n = 7) after HIVJR-FL mC inoculation. Histological data shown in C-a through -d are from a representative mouse indicated by a solid black circle, while histological data shown in C-e, -f and D are from a representative mouse demonstrated by a solid black triangle. Asterisk indicates significant difference. (P = 0.0012) (C) HIVJR-FL mc-infected cells in the lamina subserosa on day 7 after inoculation (a-d) and those in lymph nodes on day 14 after the inoculation(e and f). Panels a, c, and e show the immunostaining images of mCherry protein (in brown), while panels b, d, and f show the images of HIV-1 p24 staining(in brown). Note that infected cells are seen only in the lamina subserosa (a and b), while no infected cells were seen in lymphoid tissues (or lymph nodes) (c and d). In panels e and f, cells positive for mCherry protein and HIV-1 p24 are seen. Scale bars in magnified view denote 20 μm and all others 200 μm. (D) mCherry+ cells seen in celiac lymph nodes on day 14 after the inoculation are also positive for HIV-1 p24, CD4, and CD68. Panel a shows simultaneous staining image with anti-mCherry and anti-p24 antibodies; panel b image with anti-mCherry and anti-hCD4 antibodies; panel c image with anti-mCherry and anti-hCD68 antibodies; and panel d image with anti-mCherry and anti-hCD8 antibodies. Note that mCherry signals are seen in red while all other signals in brown. Scale bar, 20 μm.

It is noteworthy that on day 7, even in the 7 mice in which no p24 was detected in plasma, mCherry signal had been detected in all 7 mice as examined using Maestro™, suggesting that the current HIVJR-FL mC system enables to visually detect local HIV-1 replication prior to the subsequent systemic HIV-1 expansion.

3.4. Mesenteric lymph nodes are greater in number and weight in HIVJR-FL mC-exposed hNOJ mice than in HIVJR-FL mC-unexposed hNOJ mice

It should be noted that in general, no palpable lymph nodes are present in NOJ mice, which we employed in the current work (Fig. 5). However, in 5 of 6 HIVJR-FL mC-exposed hNOJ mice, readily identifiable lymph nodes were seen in the mesenteric region (Fig. 6A). The correlation between lymph node swelling and HIVJR-FL mC-exposure was significant as examined with the Fisher Exact test (P = 0.0152). We also attempted to compare the number and weight of lymph nodes in the mesenteric region of each of the 6 HIVJR-FL mC-exposed hNOJ mice on day 14 following the exposure with those of HIVJR-FL mC-unexposed hNOJ mice (n = 6). Fig. 6B shows that in the exposed mice (n = 6), the number of identified lymph nodes varied from 0 to 5; however, in the unexposed mice, no lymph nodes were identified in the same mesenteric region of the unexposed mice (n = 6)(P = 0.0025). We also excised and weighed the largest lymph node from the mesenteric region of each of the HIVJR-FL mC-exposed hNOJ mice on day 14 and those from HIVJR-FL mC-unexposed hNOJ mice after removing all surrounding tissues. Such representative tissue-free lymph nodes are shown in Fig. 6C. Fig. 6D illustrates that the weight of each largest lymph node removed from the mesenteric region of the HIVJR-FL mC-exposed hNOJ mice was greater than that from the unexposed mice (P = 0.0005).

Fig. 5. No distinct lymph nodes are present in the peritoneal cavity of NOJ mice.

Fig. 5.

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Lymphoid tissues including lymphatic vessels were seen after intraperitoneal injection of patent blue dye, by which mesenteric lymphoid tissues became readily visible. This representative picture of the abdominal area of a BALB/c mouse distinctly shows blue-stained lymphatic vessels accompanied by blood vessels and mesenteric lymph nodes as indicated by arrows (A), whereas no identified lymph nodes were in the same area in a NOJ mouse (B). Scale bars denote 1 cm.

Fig. 6. Mesenteric lymph nodes are greater in number and weight in HIVJR-FL mC-exposed hNOJ mice than in HIVJR-FL mC-unexposed hNOJ mice.

Fig. 6.

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(A) In vivo spectral fluorescence imaging enables identification of lymph nodes otherwise unidentified (or highly difficult to identify) in HIVJR-FL mC-infected humanized mice. The composite image with white light spectra and a mCherry spectrum (wave lengths 587–610 nm) demonstrates the location of the mesenteric lymph node (i-a) and iliac lymph node (i-c). Extracted mCherry spectral imaging is clearly shown in the mesenteric lymph node (i-b) and iliac lymph node (i-d). Fixed/paraffin-embedded tissue sections of the lymph nodes were immunostained with anti-hCD45 (ii-a: mesenteric lymph node, ii-d: iliac lymph node), anti-mCherry (ii-b: mesenteric lymph node, ii-e: iliac lymph node), and anti-HIV-1p24 (ii-c: mesenteric lymph node, ii-f: iliac lymph node) antibodies. Nuclei were counterstained with Mayer's hematoxylin (blue). Scale bar, 20 μm. (B) Comparison of the numbers of the mesenteric lymph nodes. The numbers of lymph nodes in the individual HIVJR-FL mC-exposed hNOJ mouse and unexposed mouse were counted by MVX10-Nuance™ with spotted mCherry signals. The numbers of lymph nodes varied from 0 to 5; however, in the unexposed mice, no lymph nodes were identified in the mesenteric region of those mice. Asterisk indicates significant difference. (P = 0.0025). (C) Representative images of the harvested and measured lymph nodes. (a) indicates a mesenteric lymph node from the HIVJR-FL mC infected mouse and (b) indicates a celiac lymph node from the HIVJR-FL mC -infected mouse, whereas (c) shows a celiac lymph node from the control mice. Scale bar, 1 mm. (D) Comparison of the weight of the mesenteric lymph nodes between non-HIVJR-FL mC-infected mice and HIVJR-FL mC-infected mice. Weight was measured using a microbalance as wet weight. We considered the weight of macroscopically undetectable mesenteric lymph node as 0 mg. Asterisk indicates significant difference. (P = 0.0005). (E) Comparison of the weight of the celiac lymph nodes between non-HIVJR-FL mC-infected mice and HIVJR-FL mC-infected mice.

We also compared the weight of lymph nodes in the celiac region of each of the HIVJR-FL mC-exposed hNOJ mice on day 14 and HIVJR-FL mC-unexposed hNOJ mice. The weight of the largest celiac lymph nodes was mostly greater in the HIVJR-FL mC-exposed hNOJ mice than in the HIVJR-FL mC-unexposed hNOJ mice, although the difference was not statistically significant (P = 0.054)(Fig. 6E).

3.5. Cell-associated virions induce more efficient & rapid transmission than cell-free virions

In the natural history of HIV-1 infection, the virus is transmitted through two modalities: (i) cell-free transmission and (ii) cell-associated transmission. We, therefore, attempted to examine whether cell-free and/or cell-associated HIV-1 are well transmitted in the present HIVJR-FL mC-hNOJ system using a protocol illustrated in Fig. 7A. Since HIV-1 is transmitted between individuals, who generally have different HLA phenotypes, we also used hPBMC from an additional HLA-A2−/A31+ healthy Individual-B in addition to those from Individual-A (HLA-A2+/A31−).

Fig. 7. Cell-associated and cell-free virions induce more efficient and more rapid transmission than cell-free virions alone in hNOJ mice.

Fig. 7.

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(A) Protocol of examination of cell-associated and cell-free HIVJR-FL mC transmission. (B) Cell-associated and cell-free virions more efficiently and more rapidly caused viremia than cell-free virions alone. Plasma samples from the HIVJR-FL mC-exposed mice were subjected to the fully automated chemiluminescent enzyme p24 immunoassay on days 7 and 14 following HIVJR-FL mC inoculation. Note that the HIVJR-FL mC viremia levels were greater in mice receiving both cell-associated and cell-free virions than in those receiving cell-free virions alone. Histological data of a solid black square is shown in panel C. (C) Distribution of HIVJR-FL mC-infected HLA-A2+ cells (Individual-A), HIVJR-FL mC-infected HLA-A31+ cells (Individual-B), and HIV-1 p24+ +and hCD68+ cells in the omentum area on day 7 after cell-free & cell associated virion exposure. Each panel demonstrates a merged image, which is composed of pictures obtained from 3-color fluorescent immunostaining. HLA-A2+ cells (a–d), HLA-A3+ cells (c, e and f), or hCD68+ cells (d) are shown in red, whereas HIV-1 p24+ cells (a–d) and HLA-A+ (e) cells are shown in green. Nuclei (a-d and f) are in blue. Panel a demonstrates the specific region where HLA-A2 & HIV-1 p24 double positive cells are located and panel b represents a magnified view of the enclosure in panel a. Panel c shows that HLA-A31+ cells (Individual-B) are also positive for HIV-1 p24. Panel d shows that HIV-1 p24+ cells are also positive for hCD68+. Panels e and f show that both HLA-A31+ and HLA-A+ cells are seen in the same area. Arrows indicate double positive cells, which are shown in yellow (a–d). Scale bar in panel c denotes 100 μmm and all others 10 μm.

Twenty-two hNOJ mice were transplanted with HLA-A2+/A31hPBMC (Individual-A), and twelve of the 22 mice were inoculated with cell-free HIVJR-FL mC virions (containing 300 ng p24), while the remaining ten mice were inoculated with the same amount of cell-free HIVJR-FL mC virions in addition to 2 x 106 HLA-A2−/A31+ PBMCs (Individual-B) that had been exposed to HIVJR-FL mC over 9 days and thoroughly washed. When examined with the Maestro™ in vivo image capturing system, distinct mCherry signals were seen from inside the abdominal cavity in all of the 22 hNOJ mice exposed to the virus. Only in 3 of the ten exposed mice, moderate levels of HIVJR-FL mC viremia were seen (Fig. 4B). The same was true in the second set of the experimental data shown in Fig. 7B. However, it was noted that as assessed on day 7 following the exposure to both cell-free and cell-aassociated HIVJR-FL mC virions, greater levels of viremia were seen in 6 of the 10 exposed mice than in the mice receiving cell-free HIVJR-FL mC virions alone (positivity, 4 of the 12 mice examined). The difference in the magnitude of viremia between the 4 cell-free virion-exposed mice with viremia (shown as 4 closed circles in Fig. 7B) and the 6 cell-free and -associated virion-exposed mice with viremia (shown as 5 closed triangles and 1 square in Fig. 7B) was 16-fold (geometric means: 894 and 15,059, respectively). When we compared all the p24 levels in the plasma of the mice on day 7 following the inoculation, the specimens from the mice receiving both cell-free and cell-associated virions showed significantly greater levels of p24 than those from the mice receiving cell-free virions alone (P = 0.008)(Fig. 7B). When the plasma p24 levels were determined on day 14 following the exposure, there was no significant difference between the two mice groups. These data strongly suggest that the addition of cell-associated virions resulted in significantly more efficient and rapid HIV-1 transmission and propagation. In the mice inoculated with cell-free virions plus HIVJR-FL mC-infected PBMC (Individual-B), HLA-A2+/A31− and p24+ cells were identified in the abdominal lymph nodes of those mice as early as day 7 after the inoculation (Fig. 7Ca-Cb). It was of note that in the mice inoculated with cell-free-virions alone, no infected cells were identified in all the lymph nodes examined on day 7 following the viral exposure except in the subserosa region (Fig. 4Cc-Cd). It was also noted that infected HLA-A2−/A31+ cells (Individual-B) were present in the mesenteric lymph nodes (Fig. 7Cc). When doubly-immunostained, such cells were found to be strongly positive for HIV-1 p24 and hCD68, indicating that the cells were producing an abundant amount of HIV-1 virions and were of the monocyte derived Mϕ/DC lineage (Fig. 7Cd). It was also found that both HLA-A2+/A31− (Individual-A) and HLA-A2−/A31+ (Individual-B) cells were present in the lymph nodes examined (Fig. 7Ce-Cf), strongly suggesting that Individual-A cells and Individual-B cells cohabitated in the same lymph node and probably immunologically activated each other.

4. Discussion

Combined antiretroviral therapy or cART has had a significant impact on HIV-1 infection and AIDS. Recent analyses have revealed that mortality rates of HIV-1-infected individuals have become close to that of general population (Lohse et al., 2007; Edmonds et al., 2011). Moreover, an increase in the number of patients receiving cART has brought about more than 35% decline in the number of newly infected individuals in developing countries including Sub-Saharan nations (UNAIDS report on the global epidemic 2013. http://www.unaids.org/en/resources/campaigns/globalreport2013/globalreport). Nevertheless, in the United States only, as many as 50,000 people still become newly infected each year and a range of social and economic factors places certain individuals at increased risk for HIV infection (CDC Fact Sheet: Today's HIV/AIDS epidemic 2016. https://www.cdc.gov/nchhstp/newsroom/docs/factsheets/todaysepidemic-508.pdf). Thus, PrEP, a way for those who are HIV-1-negative but are at high risk of being infected to prevent HIV-1 infection, has been considered to be important. PEP, a way to prevent HIV-1 infection after a possible recent exposure by taking ART, has also been considered to be critical. However, identifying potential antiretroviral agents for PrEP and PEP and proving that such agents are effective in preventing new HIV-1 infection in clinical trial settings are challenging especially since experimental HIV-1 infection is not possible at all and clinical trials for the proof of effectiveness require a large number of those at risk and long-term observation to get the results. Thus, animal models for the proof of utility of potential agents are needed.

The present work represents to the best of our knowledge the first report that HIVJR-FL mC-exposed hNOJ mouse model and human target CD4+ T lymphocytes and macrophages/monocytes, but not SIV or simian target cells, was established. HIVJR-FL mC had significant replication capacity in the test tube and in hNOJ mice and allowed us to readily identify otherwise unidentifiable HIVJR-FL mC replication sites in the early phase of HIV-1 infection. We also showed that as early on as day 7, HIVJR-FL mC replication sites were identifiable in the abdominal cavity (the omentum region) before systemic viremia. Furthermore, we demonstrated that HIVJR-FL mC virions plus cell-associated virions induce more efficient and rapid transmission than cell-free virions alone in our mouse model.

In the actual sexual transmission of HIV-1, in general, both cell-free and cell-associated HIV-1 virions are involved (Pudney et al., 1992; Louissaint et al., 2012). Cell-associated HIV-1 transmission is reportedly 10-fold to 1000-fold more efficient than cell-free transmission in vitro (Anderson, 2014; Chen et al., 2007; Mazurov et al., 2010). During sexual HIV-1 transmission, the virus crosses through breaks in the mucosal epithelial barrier to susceptible target cells in the lamina propria and the founder population expands locally. Local HIV-1 expansion then leads to ensuing viral dissemination into the draining lymph nodes, and subsequent entry to the bloodstream occurs, establishing self-propagating infection in secondary lymphoid tissues (Pope and Haase, 2003; Haase, 2010; Alford et al., 2009; Bomsel, 1997). The magnitude and rapidity of transmission and propagation of cell-associated HIV-1 have been shown to be much greater than those of cell-free HIV-1 in small animal models; however, analysis of HIV-1 transmission and propagation in the two different modes has been attempted only in a few studies (Moench, 2014; Salle et al., 2010), but such endeavors have been mostly rather unsuccessful. As shown in Fig. 7B, as assessed on day 7 following exposure to both cell-free and cell-aassociated HIVJR-FL mC virions, greater levels of viremia were seen in those mice than in the mice receiving cell-free virions alone. The difference in the magnitude of viremia between the cell-free virion-exposed mice and the cell-free and -associated virion-exposed mice was 16-fold, strongly suggesting that the addition of cell-associated virions resulted in significantly more efficient and rapid HIV-1 transmission and propagation. It was also noted that hPBMC from two donors coexisted in the same propagation sites (Fig. 7C), which may suggest a possibility that cells from a donor also cross through breaks in the mucosal epithelial barrier and immunologically activate the cells of a recipient in the initial HIV-1 expansion sites, as well as in the further established HIV-1 propagation sites, thus leading to highly efficient HIV-1 transmission and propagation in the actual sexual HIV-1 transmission.

In conclusion, the present data show that visualization of HIV-1 virions with mCherry fluorescent protein helps examine initial phases of HIV-1 transmission and propagation in hNOJ mice. The present HIVJR-FL mC-exposed hNOJ mouse model should be of utility in further study of early phases of HIV-1 transmission and spreading, as well as in the evaluation of the effects of experimental antiretroviral agents and in particularly, in the settings of PrEP and PEP.

Supplementary Material

Supplemental data

Acknowledgments

The authors thank Y. Yonemura, R. Kariya, and K. Maeda for valuable comments and suggestions. The present work was supported in part by a Grant for Development of Novel Drugs for Treating HIV-1 Infection and AIDS from Japan Agency for Medical Research and Development, Grants from Japan Society for the Promotion of Sciences, a Grant from National Center for Global Health & Medicine, and a Grant from the Intramural Research Program of Center for Cancer Research, National Cancer Institute, National Institutes of Health.

Footnotes

Conflict of interest

The authors declare no conflict of interest.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.antiviral.2017.03.027.

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