The AIDS pandemic, caused by HIV-1, continues to escalate worldwide. Although advances have been made recently in the development of antiviral drugs, a safe and efficacious AIDS vaccine is still needed urgently. The SIV/macaque model has been used widely to evaluate safety and efficacy of candidate AIDS vaccines. Pathogenic SIV induces clinical disease patterns that are typically seen in HIV-1 infected individuals, such as decrease of CD4 T-cell counts, wasting, autoimmune disorders, opportunistic infections, and malignancies . Initially, live attenuated SIV vaccines were considered the most promising approach to achieve protection against infection with pathogenic SIV ([2–8], for a review see ). However, in subsequent efficacy studies, no protection against infection with heterologous viruses was found . Furthermore, serious safety problems arose. Several monkeys vaccinated with SIVmacC8, a molecularly cloned SIV strain with a 12-base pair in-frame deletion in nef  and used in European vaccine trials, had unexpected rises in viral loads followed by disease progression . Genetic analysis of the vaccine strain revealed restoration of the nef deletion; the nef gene gradually evolved to resemble the amino acid sequence of the parental virus clone SIVmacJ5 . Such reversions to full-length, intact nef reading frames have been observed independently by several groups [8,12,13].
We reported persistent infection and pathogenicity after vaccination of rhesus macaques with SIVmac239Δ3 [14,15], a nef-deleted vaccine strain that had been further attenuated by additional large deletions in vpr and the long terminal repeat that overlaps with the remaining nef coding region and the negative regulatory element . Most monkeys vaccinated as infants with SIVmac239Δ3 progressed to AIDS ; one adult transiently immunosuppressed with steroids during vaccination also developed AIDS [14,15]. Analysis for viral genotypes of the vaccine strain by PCR revealed additional deletions in the upstream region of the long terminal repeat [14,15], as also reported by others . We noted a striking association between disease progression and the emergence of shorter viral genomes [14,15]. The additional deletions led to restoration of an open, albeit truncated Nef reading frame (unpublished data). Similar observations and expression of a novel truncated Nef protein were also reported in SIV constructs containing either a large nef deletion or interleukin-2 in place of nef .
In our earlier study , six out of eight infants vaccinated with SIVmac239Δ3 succumbed to AIDS, a sufficiently large number to assess median survival, which was approximately four times longer than those of infants infected at birth with wild-type SIVmac251. Although the vaccine strain was pathogenic in most infants, disease progressed more slowly as compared to infants infected with wild-type virus. The question remained whether SIVmac239Δ3 would exhibit a similar pattern in adult vaccinees. Here, we demonstrate that the majority of adult monkeys vaccinated with SIVmac239Δ3 developed chronic, recurrent viremia during long-term follow-up of up to 6.8 years. We observed decreased T-cell counts in all animals with high virus loads and cytoviremia, some of which progressed to AIDS.
Materials and methods
Animals, animal care and experimental design of the study
Eleven adult rhesus monkeys (Macaca mulatta) were followed in this long-term study. We also provide follow-up data of three animals vaccinated before or at birth [14,15]. The animals were housed according to NIH Guidelines for the Care and Use of Laboratory Animals at the Yerkes National Primate Research Center, Emory University (Atlanta, Georgia, USA). This facility is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Animal experiments were approved by the Animal Care and Use Committee of this institution and the Dana-Farber Cancer Institute. All animals were negative for simian T-lymphotropic virus type I  and simian retrovirus type D by serology and PCR . Vaccination procedures using SIVmac239Δ3 have been described [14,15]. Here, we present data on the surviving animals; details are given in Tables 1 and 2. Some monkeys were removed from the study before developing disease because of enrollment into a challenge study.
Virus isolation and determination of viral RNA loads
Co-cultivation of peripheral blood mononuclear cells (PBMC) has been described . Viral RNA loads were measured in early samples with a branched DNA assay . For follow-up samples and for some early samples anticoagulated with sodium citrate or EDTA, we used a more sensitive, one-tube real-time reverse transcription (RT)–PCR that was based on a fluorogenic probe and that had a lower detection limit of 50 RNA copies/ml plasma . Recurrent persistent viremia was defined by positive RT–PCR on at least three occasions (usually at least 2–3 months apart).
Plasma samples were analyzed for the presence of specific antibodies [21,23] using commercially available Western blot strips prepared from HIV-2 antigens (Cambridge Biotech Corp., Rockville, Maryland, USA), which had been previously shown to cross-react with SIV antisera.
In situ hybridization
Productively infected cells were localized in formalin-fixed, paraffin-embedded sections of lymph node, spleen, and gut by in situ hybridization for SIV RNA, as described previously . Prepared tissue sections were hybridized with a digoxigenin-labeled antisense riboprobe which spans the entire genome of the SIVsmmPGm5.3 molecular clone of SIV (Loffstrand Labs, Gaithersburg, Maryland, USA). Bound probe was detected by immunohistochemistry, using an alkaline phosphatase-conjugated anti-digoxigenin monoclonal antibody and the chromagen NBT/BCIP (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate; Roche Diagnostics Corp., Indianapolis, Indiana, USA).
We previously demonstrated that a live attenuated SIVmac239Δ3 vaccine induced AIDS in six of nine infant and in one of 16 adult monkeys . The single adult had been immunosuppressed at the time of vaccination . Follow-up times ranged from 0.6 to 4.5 years for infants and from 1.6 to 5.2 years for adults. Three out of 11 adults that were still alive at that time had developed recurrent persistent viremia and eight had early signs of immune suppression, such as persistently inverted CD4 : CD8 T-cell ratios or low CD4CD29 T-cell counts . We monitored the 11 adult macaques for another 3 years (up to 6.8 years after vaccination). We also provide additional follow-up on the three surviving monkeys exposed to the vaccine either as fetus or as neonates.
Recurrent, chronic viremia in adult vaccinees: a poor prognostic sign
When adult monkeys were vaccinated with SIVmac239Δ3, viremia generally resolved after acute infection. No vaccine-related disease was reported by Wyand et al.  in the vaccine trial in which the animals were challenged with wild-type SIVmac251 after a maximal time of 79 weeks. However, during prolonged follow-up of up to 6.8 years, we found that the majority of adult vaccinees (68%) developed chronic, recurrent viremia as measured by RT–PCR (Table 1). In two animals, plasma viral RNA loads have thus far remained low (1 × 102 copies/ml) (Table 1). In these animals, no infectious PBMC were found when 1 × 106 cells were sampled. All other animals with chronic recurrent viremia had much higher viral RNA loads (values ranging from 1 × 103 to 1 × 106 copies/ml; Table 1), which was associated with cytoviremia in all cases.
When the adult vaccinees were stratified according to the presence or absence of recurrent viremia (as measured by viral RNA load) and cytoviremia, a pattern emerged. All animals with measurable cytoviremia and viral loads ≥ 1 × 103 copies/ml developed decreased CD4 T-cell counts (Fig. 1a, Table 1). Two animals progressed to AIDS as defined by persistent CD4 T-cell counts < 200 × 106/l. In contrast, virus isolation-negative animals have so far maintained CD4 T-cell counts > 500 × 106/l, with one exception (Fig. 1b and c).
The lowest CD4 T-cell counts at the end of the observation period were found in the monkeys with persistent cytoviremia (Fig. 1a). The counts ranged from 173 × 106 to 389 × 106 cells/l (median, 268 × 106 cells/l). The age of the animals was between 8.0 and 13.5 years (median, 10.3 years). In monkeys that had been virus-isolation negative (Fig. 1b and c), final CD4 T-cell counts ranged from 405 × 106 to 1116 × 106 cells/l (median, 794 × 106 cells/l) with the age of the monkeys ranging between 7.5 and 12 years (median, 9.5 years). We found significant differences in the CD4 T-cell counts between these two groups: virus-isolation positive monkeys had significantly lower CD4 T-cell counts than virus-isolation negative animals (Mann–Whitney U-test, P = 0.0043), while the two groups did not significantly differ in age or duration of infection. We also compared the SIVmac239Δ3-vaccinated monkeys with six unvaccinated, age-matched controls, which were kept at the same primate center. The CD4 T-cell counts of the latter six animals ranged from 439 × 106 to 1463 × 106/l (median, 865 × 106 cells/l); the age range was 10.3–12.3 years (median, 12.1 years). Again, we found significantly lower CD4 T-cell counts in the virus isolation-positive monkeys compared to the unvaccinated controls (Mann–Whitney U-test, P = 0.0043), while the age of the animals was not significantly different. The CD4 T-cell counts of the virus isolation-negative monkeys did not differ significantly from those of the unvaccinated controls.
We used the CD4 T-cell data to determine the probability of an adult vaccinee maintaining CD4 T-cell counts > 500 × 106 cells/l (Fig. 2); the Kaplan–Meier method was used. The resulting curve clearly differs from that of a safe vaccine, which should produce a straight line, indicating no drop in CD4 T-cell counts. The median time to declined CD4 T cells < 500 × 106 cells/l in adult vaccinees was estimated to be 5.2 years (Fig. 2). This period is longer than that described in other studies for wild-type SIVmac239, where 50% of the rhesus monkeys died with characteristic SIV-induced immunodeficiency disease within 1 year  or for SIVmac251, where four out of four infected rhesus monkeys died with decreased CD4 T-cell counts and development of AIDS within 8 months of intravenous infection . Thus, the SIVmac239Δ3 vaccine strain is pathogenic in adult macaques; however, the disease progression rate is slower than with wild-type viruses.
AIDS in adult vaccinees
Two macaques given SIVmac239Δ3 as healthy, immunocompetent adults developed AIDS with CD4 T-cell counts < 200 × 106 cells/l after 6 and 6.6 years of follow-up, respectively. Although opportunistic infections were not observed, both animals had histological changes that are often seen in SIV-infected animals. Monkey NV6 was sacrificed due to extensive weight loss at week 312 (Table 1). Significant findings at necropsy included anemia and endometriosis. Histological evaluation of lymphoid tissues revealed changes compatible with SIV infection, including follicular hyperplasia and paracortical expansion of most lymph nodes and similar changes in the spleen. Lymphoid aggregates were observed in the bone marrow and renal interstitium, similar to lesions that have been described previously in SIV-infected animals . In situ hybridization for viral RNA revealed productively infected cells in the paracortices of lymph nodes and in the white and red pulp of the spleen (data not shown).
The second animal that developed AIDS, 6VJ, showed anorexia, anemia and frequent urinary tract infections as well as dysmenorrhea. The monkey experienced substantial weight loss with the onset of persistent diarrhea (campylobacteriosis) (Table 1). Histological evaluation upon necropsy at week 343 revealed changes characteristic of SIV infection, which included marked lymphoid hyperplasia (spleen, gut-associated lymphoid tissue, lymph nodes) and lymphoid aggregates in the kidney, bone marrow, and liver. In addition, the animal had severe, chronic active colitis. In situ hybridization revealed large numbers of productively infected cells in the white pulp of the spleen and in the paracortical zones and germinal centers of lymph nodes (data not shown).
Follow-up of monkeys vaccinated during fetal or neonatal life
All three monkeys vaccinated with SIVmac239Δ3 in the perinatal period showed signs of immune dysfunction (Table 2). At the end of the experiment, the three animals were between 5.3 and 7.1 years of age (median, 6.3 years) and had CD4 T-cell counts between 88 × 106 and 372 × 106 cells/l (median, 357 × 106 cells/l). The three monkeys had significantly lower CD4 T-cell counts (Student's t test with Welch's correction for unequal standard deviations; P < 0.0001) than a group of 10 unvaccinated clinically healthy rhesus monkeys . The latter group of animals had between 657 × 106 and 2923 × 106 CD4 T cells/l at the age of 5.8–7.8 years . One of the SIVmac239Δ3-vaccinated monkeys was persistently viremic during late follow-up, and a second animal developed recurrent viremia (Table 2).
Remarkably, animal 95-11, vaccinated orally as a neonate, remained mostly virus isolation and RT–PCR negative after the resolution of primary viremia (Table 2). Nevertheless, 95-11 developed decreased CD4 T-cell counts < 500 × 106 cells/l (Fig. 1d, dotted line). The aviremic monkey showed also intermittently low CD4CD29 T-cell subsets, anemia, thrombocytopenia, and loss of anti-Gag antibodies (Table 2, Fig. 3). The latter heralds the development of AIDS in SIV-infected rhesus macaques and in HIV-1 infected humans .
The monkey with recurrent viremia, 95-10, has not developed overt clinical disease, despite having immunological AIDS (defined as persistent CD4 T-cell reduction to < 200 × 106 cells/l).
Monkey 93-4 became, after a brief virus isolation-negative period, persistently viremic, developed decreased CD4 T-cell counts (Fig. 1d, Table 2) and loss of anti-Gag antibodies (Table 2, Fig. 3). The animal experienced intermittent diarrhea and weight loss. Upon necropsy at week 329, emaciation, splenomegaly, mesenteric and colonic lymphadenopathy, and colitis were noted. Histological evaluation revealed chronic active colitis with multifocal granulomatous inflammation (mycobacteriosis) and multinucleated SIV-positive giant cells throughout the intestinal lamina propria and Peyer's patches (data not shown), lesions that are pathognomonic of SIV infection.
Much effort has gone into developing a safe and effective vaccine to control the HIV-1 pandemic . One of the approaches explored focused on live attenuated virus vaccines, which typically have the advantage of inducing a broad and long-lived immune response via a brief replication phase. However, in the case of HIV-1, substantial risks might originate from this replication phase, the subsequent integration of the retroviral genome into the host genome and the possible residual potential of the vaccine or its progeny viruses to induce immune suppression and AIDS after prolonged incubation. The latter is difficult to foresee and usually cannot be assessed in humans.
Studies in the SIV/rhesus macaque model, which is generally accepted for AIDS vaccine studies, clearly showed that live attenuated SIVmac239Δ3 is pathogenic in neonatal rhesus monkeys [14,15,32]. Subsequently, recurrent viremia, manifestations of immune dysregulation, and overt disease were also demonstrated in some adult SIVmac239Δ3 recipients, one of which eventually died as a result of AIDS . Pathogenicity and disease progression were also reported by other groups studying various live attenuated lentiviral vaccines in primates [7,8,13,18,33–37].
In the present study, we demonstrate that the live attenuated SIVmac239Δ3 vaccine is – given enough time – pathogenic in most adult vaccinated monkeys and not just in a low proportion or in animals vaccinated as infants. We observed signs of disease progression in adult vaccinees typical for pathogenic lentivirus infections, such as persistently inverted CD4 : CD8 T-cell ratios, decreases in absolute CD4 T-cell counts, low CD4CD29 memory T-cell subsets, thrombocytopenia, weight loss, anemia, and opportunistic infections. Recurrent viremia with cytoviremia was a poor prognostic sign: animals in this group were found to have declines in their CD4 T-cell counts, and two of them developed AIDS after prolonged observation. Of note, declining CD4 T-cells counts were seen even in an SIVmac239Δ3-infected monkey (95-11) that showed no persistent recurrent viremia. Declining CD4 T-cell counts in the absence of viremia were also reported in a person who had been infected with nef-deleted HIV-1 . Our results in SIVmac239Δ3-infected macaques indicate that live attenuated lentivirus vaccines can retain or regain the ability to induce AIDS, even when used in adult immunocompetent vaccine recipients, and moreover that the disease-inducing potential might only become evident many years after vaccination of the recipient.
Increased pathogenicity was also observed in humans after prolonged infection with nef-deleted HIV-1 [38–40]. Human long-term non-progressors of the Sydney Blood Bank Cohort, which were recipients of blood infected with nef-deleted HIV-1, remained asymptomatic between 14 and 18 years without anti-retroviral therapy [40,41]. However, subsequently the blood donor and two out of five surviving recipients showed recurrent viremia and significant loss of CD4 T cells . The blood donor developed AIDS with opportunistic central nervous system infections . HIV-1 strains isolated from long-term non-progressors infected with nef-deleted HIV-1 had changes similar to those observed in virus isolated from SIVmac239Δ3-vaccinated monkeys, such as additional deletions in the nef gene [15,42,43]. Thus, long-term safety studies of attenuated vaccine viruses in the SIV/rhesus macaque model can generate data that closely resemble the situation in human infection with similar viruses.
Long-term studies, however, are very time consuming and costly. Earlier, we suggested neonatal macaques for faster and rigorous safety testing of candidate vaccines [15,42,43]. The present observations underline the relevance of the neonatal model, as results obtained from infants within relatively short periods were mirrored by the outcome in adults after long-term follow-up. Alternatively, rapid viral passages could be used for safety testing of candidate live attenuated virus vaccines. Recently, we demonstrated development of AIDS in a rhesus macaque after prolonged infection with SHIV-vpu+ , a simian-human immunodeficiency virus, which encodes env of the laboratory-adapted HIV-IIIB. Sequence analysis of the env gene and characterization of co-receptor use and cell-tropism revealed changes similar to those described after rapid passage in rhesus and pig-tailed macaques of chimeric viruses encoding IIIB env [45–50] and in a laboratory worker who progressed to AIDS after accidental HIV-IIIB infection . Thus, viral evolution proceeded similarly in chronically infected individuals during disease progression as well as after serial virus passage .
SIVmac239Δ3 is not only pathogenic in most adult macaques after prolonged follow-up, as shown in the present study, but also had only limited efficacy as a vaccine. When four SIVmac239Δ3-vaccinated monkeys were challenged with heterologous, pathogenic SHIV89.6P, all became infected, although they did not develop acute disease . Among another four SIVmac239Δ3-vaccinees that were challenged with uncloned, heterologous, pathogenic SIVsmE660 28 months after vaccination, all became infected, and three had to be sacrificed because of T-cell depletion and clinical deterioration approximately 1 year post-challenge . Moreover, we recently found live attenuated SIVmac239Δ3 to be ineffective against homologous virus challenge performed 5 years post-vaccination. All three SIVmac239Δ3-vaccinated animals (95-11, 6543, N437) that were challenged intravenously with SIVmac251 (as control animals for another study) were superinfected with challenge virus; one monkey subsequently developed AIDS 15 months post-challenge (unpublished data). Thus, live attenuated SIVmac239Δ3 induced neither broad nor long-lived immune responses.
The question remains whether live attenuated HIV-1 vaccines with high efficiency and low risk of AIDS induction will be attainable [9,35,42,52–56]. Thus far, deletion mutant viruses with almost every desired level of attenuation have been constructed ; however, the efficacy of protection was inversely related with the degree of attenuation . This is in accordance with the ‘threshold hypothesis': an attenuated virus must replicate at a sufficiently high rate to trigger protective immune response [9,14,58,59]. More recently, live vaccine viruses were constructed with various, often multiple deletions, including removal of potential env deglycosylation sites; live attenuated viruses have also been combined with other immunogens [60–65]. Long-term safety and efficacy of the various vaccines and vaccine regimes await further testing.
Recently, the tradeoff between HIV-1 vaccine efficacy (in terms of preventing new infections) and safety problems (in terms of vaccine-induced AIDS deaths) was estimated in a theoretical mathematical model . The authors predicted that in countries with high HIV-1 transmission rates, a mass vaccination campaign with live attenuated HIV-1 vaccines would significantly decrease the AIDS death rate. This calculation was based upon the assumption that vaccines would have fairly high efficacy (50% to 95% protection against infection), and that the vaccine would cause 1% to 10% (most likely value of 5%) of vaccinated individuals to progress to AIDS within 25 years . These assumptions were overly optimistic and clearly are not compatible with our observations in rhesus macaques vaccinated with the multiply deleted SIVmac239Δ3. New data show lack of efficacy and much more serious safety problems with this live attenuated virus vaccine than assumed previously. Thus, the safety and efficacy estimates that formed the basis of the mathematical model need to be revised to reflect the more sobering data on live attenuated, nef-deleted vaccine candidates.
We thank S. Sharp for the preparation of this manuscript.
Sponsorship: This work was supported in part by National Institutes of Health grants RO1 RR14180 awarded to R.M.R., and the Yerkes Center base grant RR00165 to H.M.M., D.C.A., and S.O. Also supported by the Center for AIDS Research core grant IP3028691 awarded to the Dana-Farber Cancer Institute as support for AIDS research efforts, and by the Swiss National Science Foundation grant 823A-50315 to R.H.-L.
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