Inoculation with Cell-Free Human Immunodeficiency Virus ... treatment of human immunodeficiency virus type 1 infection would be a valuable resource for AIDS ...
JOURNAL OF VIROLOGY, Sept. 1993, p. 5367-5374
Vol. 67, No. 9
0022-538X/93/095367-08$02.00/0 Copyright © 1993, American Society for Microbiology
Serological, Biological, and Molecular Characterization of New Zealand White Rabbits Infected by Intraperitoneal Inoculation with Cell-Free Human Immunodeficiency Virus SALVATORE REINA,1'2 PHILLIP MARKHAM,2 ELISABETH GARD,3 FOUAD RAYED,3 MARVIN REITZ,2 ROBERT C. GALLO,2 AND OLIVIERO E. VARNIERlt* Laboratory of Human Retrovirology, Institute of Microbiology, School of Medicine, University of Genoa, 16132 Genoa, Italy'; Laboratory of Tumor Cell Biology, National Cancer Institute, Bethesda, Maryland 208922; Advanced Bioscience Laboratories, 5510 Nicholson Lane, Kensington, Maryland3 20895 Received 22 February 1993/Accepted 6 May 1993
The availability of a small laboratory animal model suitable for the evaluation of methods for prevention and treatment of human immunodeficiency virus type 1 infection would be a valuable resource for AIDS research.
Here we describe the infection of a strain of domestic rabbits by intraperitoneal inoculation with cell-free human immunodeficiency virus type 1. Evidence of infection includes the presence of an immune response that has persisted for almost 3 years and the detection of and reisolation of infectious virus from peripheral blood mononuclear cells (PBMCs) and other tissues during the first 2 years. Typical viral proteins, DNA and RNA patterns, were observed in rabbit PBMCs and in cells infected by cocultivation with rabbit PBMCs. While a number of possible pathological changes were evaluated in infected rabbits, the presence of changes in lymph node structure similar to those reported in infected humans merits further investigation. human lymphoid tissues, which can then be infected in vivo by HIV-1 (18, 25, 30), are being evaluated (6). However, as with any model system, these approaches are artificial and, particularly with mice, are subject to induction of interactions with endogenous murine viruses which may further complicate the interpretation of experimental observations. Several investigators have reported that the domestic rabbit can, with appropriate manipulation, be infected by human retroviruses including both human T-cell leukemia virus type I (HTLV-I) and HIV-1 (2, 8, 20, 21, 40). The transmission of HTLV-I to the domestic rabbit is well documented and in many cases occurs in a manner similar to that observed in humans, i.e., transmission by blood and semen and transmission from mother to offspring in the uterus or in milk (15). While no consistent pathological consequences of infection of the rabbit by HTLV have been reported to date, the model is being used for the evaluation of methods to prevent and treat infection (26, 32, 38). We and others have previously reported the infection of domestic rabbits by HIV-1, including infection of naive rabbits as well as rabbits previously infected with HTLV-I (39). While those studies primarily used HIV-1-infected cells, preliminary results also suggested that cell-free virus could also be used to produce infection (8). The purpose of the studies reported here is to confirm and further document the reproducible and productive infection of the domestic rabbit by cell-free HIV-1 and to serologically, biologically, and molecularly characterize infected rabbits and virus
An important priority in AIDS research is the development of an animal model that can be used to study infection and pathogenesis as well as to evaluate methods of prevention and treatment of human immunodeficiency virus type 1
(HIV-1) infection (4, 5, 6, 11, 19, 27, 41). The ability of simian immunodeficiency viruses to infect several species of macaque has provided important insights into infection and pathogenesis by this group of retroviruses (7, 29, 37). The related human retrovirus HIV-2 can also infect some species of nonhuman primates, although infection occurs without reproducible pathological effects (9, 31, 35). Limitations of these models include their expense and the need for highly specialized housing facilities. Questions also exist regarding the suitability of using these viruses, which, although related, are clearly distinct from HIV-1 and therefore cannot be used to study the characteristics of HIV-1. Animal models to directly study HIV-1 have also had their limitations. Two higher species of primate, the chimpanzee (24) and gibbon ape (23), and more recently the macaque species Macaca nemestrina (1) have been reported to be susceptible to infection by HIV-1. It is not yet clear whether symptoms of an AIDS-like disease occur in these animals. In the chimpanzee, HIV-1 does induce both humoral and cell-mediated immunity, including the production of neutralizing antibodies, and this model has been extensively used to evaluate procedures for prevention or treatment of infection by HIV-1 (3, 4, 10, 14).
While much useful information has been obtained using primates, a less expensive, more available, and more easily manipulated small laboratory animal model could be a valuable asset in AIDS research. Some studies in mice, including reports of the creation of transgenic animals carrying complete copies of the HIV-1 genome (22) and the use of immunodeficient mice (SCID mice) reconstituted with
recovered from these rabbits.
MATERUILS AND METHODS Animals. Three- to 4-kg, pasteurella-free New Zealand White laboratory rabbits, obtained from Hazelton Research Products, Denver, Pa., were housed in individual cages in a biosafety level 3 facility. Prior to the experimental infection, all animals were tested for antibody to HIV-1, by enzymelinked immunosorbent assay (ELISA) and Western immu-
Corresponding author. Present address: Laboratory of Virology, AIDS Center San Luigi, H. S. Raffaele, Via Stamira D'Ancona 20, 20127 Milan, Italy. *
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FIG. 1. Schematic history (35 months) of 3 rabbits inoculated i.p. with cell-free HIV-1. Arrows indicate inoculation times. The absorbance (Ab) panel indicates optical density (O.D.) values of HIV-1 antibodies measured by ELISA in rabbit serum samples, whereas the bottom panel shows the antibody specificity confirmed by Western blot (+, positive; -, negative, according to HIV-1 Western blot positivity). Results of virus isolation are depicted in the upper panel, where A refers to the positive in vitro virus isolation from tissues of rabbit C2011. SP, spleen, LN, lymph nodes. Proviral sequences were amplified, by polymerase chain reaction (PCR) from rabbit peripheral blood lymphocytes (PBL) and necropsy tissues (B). LN, lymph nodes; Lug, lung; Liv, liver; TG, thyroid gland.
noblot, and were surgically declawed. All protocols used preapproved by the Institution Animal Welfare and Use Committee and the Biohazard Committee at Advanced BioScience Laboratories, Inc., Kensington, Md. Virus preparation. Supernatant fluids of H9 cells chronically infected by HIV-1IIIB were used either undiluted (lx) or following concentration by banding in sucrose and pelleting by high-speed centrifugation. HIV-lsc was obtained from infected supernatant fluids from activated normal human peripheral blood mononuclear cells (PBMCs) and was used undiluted at 1 x . When supernatant fluids were used to propagate virus from cell culture to fresh cell culture, cells were pelleted by centrifugation at 450 x g for 10 min and the cell-free fraction was collected. The infectious titer of these virus stocks, assayed on activated human PBMCs, was approximately 107 50% tissue culture infectious doses for concentrated HIV-1IIIB and approximately 105 50% tissue culture infectious doses for unconcentrated HIV-111IB and were
HIV-lsc. Inoculations. Eight rabbits were treated to induce aseptic peritonitis by intraperitoneal (i.p.) injection of 4% thioglycolate preparation as previously described (8). After 48 h, 5 ml of phytohemagglutinin (PHA) (10 ,ug/ml)-interleukin-2 (20 U/ml) solution was injected. Two days later, three rabbits (C2011, C2019, and C2021) were inoculated i.p. with 5 ml of 1X HIV-1IIIB and three other animals (C2010, C2022, and C2023) were inoculated with 5 ml of lx HIV-1sc. Two animals (C2020 and C2029), used as controls, were injected with 5 ml of cell growth medium. All animals were reinoculated i.p. 2 months later. Animals that had been inoculated with 5.0 ml of concentrated HIV-1111B and HIV-lsc received 5.0 ml of 1,OOOx HIV-1IIIB and lx HIV-1sc, respectively. Cell preparation. Rabbit PBMCs were prepared from heparinized blood, obtained by venipuncture from the middle vein of the ear, and by separation on Ficoll-Hypaque. The number of contaminating erythrocytes and platelets was further reduced by osmotic shock, i.e., medium was diluted with sterile H20 for 30 min and rebanded in Ficoll-Hypaque. Peripheral blood lymphocytes were also prepared from human cord blood by these procedures.
Virus isolation. Rabbit PBMCs were stimulated by overnight incubation in growth medium (RPMI 1640, 15% fetal calf serum, 1% nonessential amino acids [GIBCO], 5% glutamine) supplemented with 10 ,ug of PHA (PHA-P, GIBCO) per ml. Cells were then washed with magnesiumfree phosphate-buffered saline (PBS) and incubated in growth medium containing 3.5 ng of phorbol ester myristate acetate (Sigma) per ml for an additional 1 h. These primary cell cultures were then incubated at 37°C in growth medium supplemented with 120 U of human purified interleukin-2 (TCGF; Cellular Products Inc) per ml. Rabbit cells were then carried either as primary cultures or were used in cocultivation experiments. Cells used as targets included activated human cord blood, PBMCs (prestimulated in growth medium supplemented with 5 ,ug of PHA-P per ml), or cells from established human lymphoid cell lines, such as H9, Molt-3, HUT-78, and MT-2 or the human promonocytic cell line U937. RTA. Reverse transcriptase activity (RTA) was detected by using established procedures to monitor supernatant fluids, concentrated -20-fold by precipitation with 10% polyethylene glycol as described previously. Oligo(dT)poly(A) or oligo(dT)-poly(dA) was used as primer template, and Mn2" was the cation (16). Soluble p24 antigen. HIV-1 p24 antigen was detected and quantified in unconcentrated supematant fluids by antigencapture ELISA (Organon Teknika, Durham, N.C.). Viral proteins. Radioimmunoprecipitation, Western blot, and indirect immunofluorescence assays were performed to evaluate HIV-1 protein expression in cells or cell lysates as previously described (17). Electron microscopy. The presence of HIV-1-like virions was visualized by electron microscopic examination of cells fixed in 2.5% formaldehyde and further processed as described previously (17). Southern and Northern (RNA) blots. Nucleic acids were extracted from chronically infected rabbit cell cultures, and Southern and Northern blots were performed to identify HIV-1 DNA and RNA sequences. HindIII and Sacl diges-
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tion enzymes were used to identify HIV-1IIIB isolate patterns as previously described (28). ELISA. For the detection of HIV-1 antibodies in rabbit serum or plasma, a standard ELISA, virus-lysate coated plates (DuPont de Nemours), was optimized. One hundred microliters of serum or plasma samples, diluted in PBSTween 20, was dispensed in plates and incubated overnight at 4°C. After being washed, 100 ,ul of biotinylated anti-rabbit immunoglobulin G (1:100 dilution; Vector) was dispensed and incubated for 1 h at 37°C. After a further washing cycle, an avidine-peroxidase complex was added and incubated 30 min at 37°C. In the last steps, the substrate ortho phenyl diamine was added and, at the end, the optical density was measured at 492 nm. Western blot. The Western blot assay was performed by
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FIG. 2. (A) Western blot analysis of sera from HIV-1-infected rabbits. Reactivity of sera from rabbits C2011, C2019, and C2021 against HIV-1 proteins was demonstrated by using whole disrupted HIV-11IB grown in Molt-3 cells. Serum from rabbit C2020 was used as negative control, and sera from seropositive human donors was used as positive controls. (B) Western blot analysis of sera from HIV-1-infected rabbits, using whole disrupted virus supplemented with recombinant HIV-1 gpl60 as antigen. Western blot strips prepared with whole disrupted HIV-1 supplemented recombinant envelope proteins were used to evaluate the reactivity of HIV-1 proteins against the sera of HIV-1H1IB-infected rabbits (C2011, C2019, and C2021). Serum from a patient with AIDS was used as a positive control, and serum from uninfected rabbit C2029 was used as a negative control.
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using nitrocellulose strips loaded with lysates of HIV-1IIIB in either Molt-3 or H9 cells according to previously described methods (34). Commercially prepared strips supplemented with HIV-1 gp160 (Epitope Inc.) were also used.
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unImmune response concentrated HIV-111IB or HIV-1sc exhibited little or no evidence of infection in the first 2 months postinoculation and were reinoculated with approximately 107 infectious units of HIV-lIIIB or again with lx HIV-1sc. These animals developed a humoral immune response to HIV-1 proteins detected by ELISA, Western blot, and radioimmunoprecipitation procedures. In the case of animals receiving concen-
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FIG. 5. RTA detected in cells infected by virus isolated from cultured primary rabbit leukocytes. RTA was analyzed in supernatant fluids from H9 cells infected with HIV-1 rabbit isolate following serial passages in fresh H9 cells exposed to cell-free virus recovered from the previous passage. Supernatant samples were collected at 2-day intervals and tested in triplicate. Bars indicate peaks of RTA detected in three cell cultures; supernatants were collected between days 15 and 20 for four generations of virus.
FIG. 3. Indirect immunofluorescence assay of HIV-1 antigens. The assay detected viral protein expression in human cord blood leukocytes, infected by HIV-1 isolated from primary rabbit PBMCs by coculture, and was performed using serum from a patient with AIDS. Magnification, x455.
trated virus, ELISA levels have persisted at moderate levels, in some instances for more than 3 years (Fig. 1). Those animals receiving an approximately 100-fold lower concentration of HIV-lsc exhibited only a low and transient immune response, usually detectable for only a few months
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FIG. 4. Radioimmunoprecipitation of HIV-1 proteins in U937 cells infected by virus from rabbit C2019. Primary leukocytes from rabbit C2019 were cocultured with cell line U937 as described in Materials and Methods. Lanes: A, cells precipitated with normal human serum; B, cells precipitated with serum from an HIV-1seropositive patient; C, culture supernatant precipitated with serum from an HIV-1-positive patient; D, molecular weight standards.
postinoculation. The specificity of this immune response was further demonstrated by Western blot analysis, indicating antibodies recognizing the HIV-1 structural proteins encoded by gag (p17, p24, and p55) and env (gp4l) genes (Fig. 2A). As shown in Fig. 2B, using Western blot strips supplemented with HIV-1 recombinant gpl60, this seroreactivity also included antibodies recognizing HIV-1 gpl20 and gpl60 proteins. The intensity of the virus-specific bands gradually increased over time as predicted by the pattern seen by ELISA. A band with a size of -31 kDa, when HIV-1 was grown in H9 cells, is nonspecific and apparently was due to reactivity with cellular proteins carried with virus isolated. This persistent level of antibodies to many HIV-1 structural proteins is consistent with that expected in chronically infected animals. To further document infection by HIV-1, attempts were made to isolate virus from inoculated animals and to characterize viral protein profiles and nucleic acids in cultured PBMCs recovered from inoculated animals or in cells used to isolate virus from these animals. Virus isolation. PBMCs from inoculated and control rabbits were prepared as mentioned above and initially propagated as primary cultures in medium supplemented with interleukin-2. Cultures showing evidence of viral expression were further cocultivated with activated human cord blood leukocytes or with one or more established cell lines, i.e., H9, HUT-78, Molt-3, or U937. Virus isolation was attempted in PBMC samples collected during the first 2 years of the follow-up period. Virus was detected in and isolated from all three rabbits infected with the highest concentration of HIV-1IIIB, beginning approximately 2 months postinoculation. The rate of virus recovery ranged between 50 and 70% (Fig. 1). As shown in Fig. 3, after 12 days of cocultivation, rabbit primary PBMCs cocultured with activated human cord blood leukocytes show the typical morphology of syncytia induced by HIV-1 infection in vitro. Viral protein expression was detected by using anti-HIV-1 human polyclonal serum and was further confirmed by radioimmunoprecipitation assay. As shown in Fig. 4, all expected viral structural and precursor proteins, e.g., gp4l, gpl20, gp160, p14, p17, and p53, were observed in infected cells. In several cell lines cocultivated with primary leukocytes
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FIG. 6. Electron micrographs of H9 cells infected by cocultivation with rabbit PBMC cultures. PBMCs from rabbit C2019 were cocultured with T-cell hine H9 and were examined by electron microscopy on day 20 postinfection. Magnification, x90,000.
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from rabbits C2019 and C2021, HIV-1 replication was demonstrated by the presence in supernatant fluids of virus infectious for target cells and of the soluble p24 viral protein, as detected by antigen capture assay (data not shown). Interestingly, RTA was not detectable, whereas viral p24 antigen was easily determined (150 to 300 pg/ml after 2 weeks in primary rabbit cell cultures). All the HIV-1 rabbit isolates propagated in human cell lines continued to exhibit minimally detectable RTA. The significance of the sporadic and low-level RTA detected in many cultures is not known but is consistent with the presence of RT-inhibitory activities (36) and the low virus titer of the rabbit isolates. As shown in Fig. 5, RTA became more readily detected in cultures infected by virus which had been serially propagated into uninfected cell lines. Target cell lines used to propagate rabbit isolate infectious progeny showed different sensitivities: the U937 macrophage-promonocyte cell line seemed to
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FIG. 7. DNA and RNA viral sequences were evaluated, comparing lysates of several rabbit cell cocultures. Primary rabbit cells obtained from rabbit C2019 were mixed with different targets: U937 (19UB), activated cord blood lymphocytes (19CA), and uninfected H9 cells (19CH). Lanes IIIB, a lysate of HIV-IIIB chronically infected H9 cells as a control of the same virus prototype inoculated into the animal.
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FIG. 8. (A) Micrograph of a lymph node section from rabbit C2011 inoculated i.p. with concentrated cell-free virus (HIV-IIIB). Disruption of the follicular architecture of the organ, although not totally obliterated, is visible. Magnification, x 100. (B) Magnification of the boxed area in panel A. The lymph node is diffusely infiltrated by histocytes in both the paracortical and medullary region, and a marked angiogenesis is present. Magnification, x 250.
be more suitable for short-term cocultivation with primary rabbit PBMCs in obtaining quick evidence of in vitro infection (>50 ,ug of p24 virus antigen within 10 days), whereas MT-2 cells yielded quantitively more virus. Electron microscopy. Electron microscopic examination further documented virus release. As shown in Fig. 6, both retroviral budding and mature particles were observed in those cultures in which rabbit isolates of HIV-1 were serially propagated. Large numbers of morphologically aberrant particles were observed, suggesting the presence of defective viruses. Viral nucleic acids. The presence of HIV-1 in primary rabbit leukocytes and in cocultured cells was confirmed by Southern blot, Northern blot, and polymerase chain reaction procedures to detect and characterize viral nucleic acids. Characteristic patterns, proviral DNA digested by SacI and HindIII restriction enzymes, and specific bands of viral RNA were identified in infected cells (Fig. 7). Proviral sequence amplification. The presence of HIV-1 proviral DNA was demonstrated by polymerase chain reaction (33) in bone marrow, brain, liver, spleen, and several lymph nodes collected from rabbit C2011, which died from complications of fur ball development in its stomach. Virus was isolated from some of these tissues (brain, spleen, and inguinal lymph node) after their cocultivation with MT-2 cells and final inoculation of culture supernatants into H9 cells (Fig. 1). Pathologic changes. As described in previous experiments (8), no persistent or consistent pathologic complications were observed in HIV-1-infected rabbits. In some animals, transient bilateral pulmonary rales and nonpurulent conjunctivitis were observed during the first few months postinfection. Also, in some animals several abnormalities, including bilateral or unilateral lymphadenopathy, hypoproteinemia and hypoalbuminemia, lymphocytosis, and elevated liver enzyme levels were intermittently observed. Of significance was the histopathologic examination of rabbit C2011, which died 4 months postinoculation. Physical examination revealed the presence of a trichobenzoar palpable in the left pyloric area and marked lymphadenopathy, leukocytopenia, and severe dehydration with lethargy. Microscopic examination of 20 formalin-fixed serial sections from multiple lymph nodes revealed irregularities such as the presence of enlarged lymphoid follicles, hyperplastic germinal centers, effacement of lymphoid follicles with involution of germinal centers, and the presence of focal angiogenesis (Fig. 8).
shows the presence of many defective viral particles and poor infectivity, which persist even when the virus is passed to permissive human cells, it is possible that rabbit cells adapted to HIV-1 selected after multiple passages in vivo could improve viral infectivity. For the virus isolation, several uninfected target cells were used and, in our hands, stimulated cord blood lymphocytes and U937 cells were the most sensitive for the in vitro viral outcome in primary cell cocultures; H9, Molt-3, and MT-2 cells were equally suitable for serial isolate propagation after the onset of the in vitro infection. We concluded that virus isolation can be best achieved by using primary rabbit lymphocytes cocultivated with activated human cord blood cells; similar sensitivity is also obtained with U937 cells. For rabbit isolate propagation, H9 and Molt-3 cells gave consistent results and the highest virus production was possible by superinfecting MT-2 cells. Rabbit cells released infectious HIV-1 which was passed to other target cells either by cocultivation or as cell-free virus. The observed poor ability to measure RTA could be consistent with the low viral titer and the presence of RT inhibitors, as postulated by other authors (36). In addition, comparisons between primary rabbit cell culture isolates and the HIV-lIIIB parental strain for the ability to trans activate on H9-389 target cells (12, 13) have shown that rabbit isolates did not trans activate in 52 h, whereas the HIV-IIIB control showed a typical spot profile (data not shown). Although it is clear that the domestic rabbit can be infected by HIV-1, concern remains regarding its suitability as an animal model of infection, pathogenesis, and for use in the evaluation of prophylactic or treatment protocols. Of relevance to these considerations, we have observed in a single animal that infection by HIV-1 involves early changes in lymph node structure reminiscent of those observed in infected humans. These observations, as well as the evaluation of virus distribution in infected rabbits, are continuing. If substantiated, the rabbit may provide an accessible and inexpensive model for at least some aspects of HIV-1
DISCUSSION
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