Michael K. Axthelm

Profound early control of highly pathogenic SIV by an effector memory T-cell vaccine

The acquired immunodeficiency syndrome (AIDS)-causing lentiviruses human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) effectively evade host immunity and, once established, infections with these viruses are only rarely controlled by immunological mechanisms. However, the initial establishment of infection in the first few days after mucosal exposure, before viral dissemination and massive replication, may be more vulnerable to immune control. Here we report that SIV vaccines that include rhesus cytomegalovirus (RhCMV) vectors establish indefinitely persistent, high-frequency, SIV-specific effector memory T-cell (TEM) responses at potential sites of SIV replication in rhesus macaques and stringently control highly pathogenic SIVMAC239 infection early after mucosal challenge. Thirteen of twenty-four rhesus macaques receiving either RhCMV vectors alone or RhCMV vectors followed by adenovirus 5 (Ad5) vectors (versus 0 of 9 DNA/Ad5-vaccinated rhesus macaques) manifested early complete control of SIV (undetectable plasma virus), and in twelve of these thirteen animals we observed long-term (≥1 year) protection. This was characterized by: occasional blips of plasma viraemia that ultimately waned; predominantly undetectable cell-associated viral load in blood and lymph node mononuclear cells; no depletion of effector-site CD4+ memory T cells; no induction or boosting of SIV Env-specific antibodies; and induction and then loss of T-cell responses to an SIV protein (Vif) not included in the RhCMV vectors. Protection correlated with the magnitude of the peak SIV-specific CD8+ T-cell responses in the vaccine phase, and occurred without anamnestic T-cell responses. Remarkably, long-term RhCMV vector-associated SIV control was insensitive to either CD8+ or CD4+ lymphocyte depletion and, at necropsy, cell-associated SIV was only occasionally measurable at the limit of detection with ultrasensitive assays, observations that indicate the possibility of eventual viral clearance. Thus, persistent vectors such as CMV and their associated TEM responses might significantly contribute to an efficacious HIV/AIDS vaccine.

Effector memory T cell responses are associated with protection of rhesus monkeys from mucosal simian immunodeficiency virus challenge

The rapid onset of massive, systemic viral replication during primary HIV or simian immunodeficiency virus (SIV) infection and the immune evasion capabilities of these viruses pose fundamental problems for vaccines that depend upon initial viral replication to stimulate effector T cell expansion and differentiation. We hypothesized that vaccines designed to maintain differentiated effector memory T cell (TEM cell) responses at viral entry sites might improve efficacy by impairing viral replication at its earliest stage, and we have therefore developed SIV protein-encoding vectors based on rhesus cytomegalovirus (RhCMV), the prototypical inducer of life-long TEM cell responses. RhCMV vectors expressing SIV Gag, Rev-Tat-Nef and Env persistently infected rhesus macaques, regardless of preexisting RhCMV immunity, and primed and maintained robust, SIV-specific CD4+ and CD8+ TEM cell responses (characterized by coordinate tumor necrosis factor, interferon-γ and macrophage inflammatory protein-1β expression, cytotoxic degranulation and accumulation at extralymphoid sites) in the absence of neutralizing antibodies. Compared to control rhesus macaques, these vaccinated rhesus macaques showed increased resistance to acquisition of progressive SIVmac239 infection upon repeated limiting-dose intrarectal challenge, including four macaques who controlled rectal mucosal infection without progressive systemic dissemination. These data suggest a new paradigm for AIDS vaccine development—vaccines capable of generating and maintaining HIV-specific TEM cells might decrease the incidence of HIV acquisition after sexual exposure.

Development and Homeostasis of T Cell Memory in Rhesus Macaque

The rhesus macaque (RM) is a critical animal model for studies of viral pathogenesis and immunity, yet fundamental aspects of their cellular immune response remain poorly defined. One such deficiency is the lack of validated phenotypic signatures for their naive and memory T cell subsets, and the resultant unavailability of accurate information on their memory T cell development, homeostasis, and function. In this study, we report a phenotypic paradigm allowing definitive characterization of these subsets and their comprehensive functional analysis. Naive T cells are optimally delineated by their homogeneous CD95lowCD28highβ7 integrinint (CD4+) or CD95lowCD28intCD11alow (CD8+) phenotypes. This subset 1) was present in blood and secondary lymph tissues, but not effector sites; 2) vastly predominated in the fetal/neonatal immune system, but rapidly diminished with postnatal age; 3) lacked IFN-γ production capability, and specific responses to RM CMV; and 4) demonstrated low in vivo proliferative activity. CD4+ and CD8+ memory subsets were CD95high, but otherwise phenotypically heterogeneous and included all IFN-γ production, RM CMV-specific responses, effector site T cells, and demonstrated high in vivo proliferative activity (∼10 times the naive subset). These analyses also revealed the RM “effector memory” subset within the overall memory population. This population, best defined by lack of CD28 expression, contained the majority of RM CMV-specific cells, was highly enriched in extralymphoid effector sites, and comprised an increasing proportion of total memory cells with age. The effector memory subset demonstrated similar in vivo proliferative activity and survival as CD28+ “central memory” T cells, consistent with independent homeostatic regulation.

Immune clearance of highly pathogenic SIV infection

Established infections with the human and simian immunodeficiency viruses (HIV and SIV, respectively) are thought to be permanent with even the most effective immune responses and antiretroviral therapies only able to control, but not clear, these infections. Whether the residual virus that maintains these infections is vulnerable to clearance is a question of central importance to the future management of millions of HIV-infected individuals. We recently reported that approximately 50% of rhesus macaques (RM; Macaca mulatta) vaccinated with SIV protein-expressing rhesus cytomegalovirus (RhCMV/SIV) vectors manifest durable, aviraemic control of infection with the highly pathogenic strain SIVmac239 (ref. 5). Here we show that regardless of the route of challenge, RhCMV/SIV vector-elicited immune responses control SIVmac239 after demonstrable lymphatic and haematogenous viral dissemination, and that replication-competent SIV persists in several sites for weeks to months. Over time, however, protected RM lost signs of SIV infection, showing a consistent lack of measurable plasma- or tissue-associated virus using ultrasensitive assays, and a loss of T-cell reactivity to SIV determinants not in the vaccine. Extensive ultrasensitive quantitative PCR and quantitative PCR with reverse transcription analyses of tissues from RhCMV/SIV vector-protected RM necropsied 69–172 weeks after challenge did not detect SIV RNA or DNA sequences above background levels, and replication-competent SIV was not detected in these RM by extensive co-culture analysis of tissues or by adoptive transfer of 60 million haematolymphoid cells to naive RM. These data provide compelling evidence for progressive clearance of a pathogenic lentiviral infection, and suggest that some lentiviral reservoirs may be susceptible to the continuous effector memory T-cell-mediated immune surveillance elicited and maintained by cytomegalovirus vectors.

Insufficient Production and Tissue Delivery of CD4+Memory T Cells in Rapidly Progressive Simian Immunodeficiency Virus Infection

The mechanisms linking human immunodeficiency virus replication to the progressive immunodeficiency of acquired immune deficiency syndrome are controversial, particularly the relative contribution of CD4+ T cell destruction. Here, we used the simian immunodeficiency virus (SIV) model to investigate the relationship between systemic CD4+ T cell dynamics and rapid disease progression. Of 18 rhesus macaques (RMs) infected with CCR5-tropic SIVmac239 (n = 14) or CXCR4-tropic SIVmac155T3 (n = 4), 4 of the former group manifested end-stage SIV disease by 200 d after infection. In SIVmac155T3 infections, naive CD4+ T cells were dramatically depleted, but this population was spared by SIVmac239, even in rapid progressors. In contrast, all SIVmac239-infected RMs demonstrated substantial systemic depletion of CD4+ memory T cells by day 28 after infection. Surprisingly, the extent of CD4+ memory T cell depletion was not, by itself, a strong predictor of rapid progression. However, in all RMs destined for stable infection, this depletion was countered by a striking increase in production of short-lived CD4+ memory T cells, many of which rapidly migrated to tissue. In all rapid progressors (P < 0.0001), production of these cells initiated but failed by day 42 of infection, and tissue delivery of new CD4+ memory T cells ceased. Thus, although profound depletion of tissue CD4+ memory T cells appeared to be a prerequisite for early pathogenesis, it was the inability to respond to this depletion with sustained production of tissue-homing CD4+ memory T cells that best distinguished rapid progressors, suggesting that mechanisms of the CD4+ memory T cell generation play a crucial role in maintaining immune homeostasis in stable SIV infection.

Cytomegalovirus vectors violate CD8+ T cell epitope recognition paradigms

Introduction CD8+ T cell responses focus on a small fraction of total pathogen-encoded peptides, which are similar among individuals with shared major histocompatibility complex (MHC) alleles. This focus can limit immune control of genetically flexible pathogens, such as HIV and SIV, because CD8+ T cells in most infected subjects do not target sequences required for pathogen fitness, resulting in viral escape. Although a vaccine capable of broadening or redirecting CD8+ T cell epitope targeting to prevent viral escape would be highly advantageous, it remains unclear whether this targeting can be diverted from its default pattern during priming. Fibroblast-adapted RhCMV/gag vectors elicit MHC class II–restricted CD8+ T cells, greatly expanding the breadth of the response. (Top) Differential inhibition of SIVgag-specific CD8+ T cells from SIV+, fibroblast-adapted RhCMV/gag vector–vaccinated, and tropism-repaired RhCMV/gag vector–vaccinated rhesus macaques by MHC-I versus MHC-II blockade. (Bottom) Responses to consecutive SIVgag 15mer peptides in the indicated animals, classified by sensitivity to MHC-I versus MHC-II blockade. Methods We used intracellular cytokine analysis to compare the epitope targeting of SIV-specific CD8+ T cell responses in rhesus macaques with controlled SIV infection or after vaccination with either conventional SIV vaccines or rhesus cytomegalovirus (RhCMV) vectors. RhCMV vectors have been associated with stringent control of SIV challenge in the absence of protective MHC alleles. Results Fibroblast-adapted RhCMV/SIV vectors elicited SIV-specific CD8+ T cells that failed to target any canonical epitopes associated with SIV infection or conventional SIV vaccination. Instead, they recognized distinct epitopes characterized by extraordinary breadth (greater than that of conventional vaccines by a factor of >3), MHC class II (MHC-II) restriction (63% of epitopes), and high promiscuity (epitopes common to most or all responses in vaccinated macaques). These unconventionally targeted CD8+ T cell responses recognized autologous SIV-infected cells, indicating that processing and presentation of the unconventional epitopes is CMV-independent. However, CMV gene expression was responsible for directing epitope specificity of CD8+ T cells during priming. The induction of canonical SIV epitope–specific CD8+ T cell responses was specifically suppressed by expression of the Rh189/US11 gene, and the promiscuous MHC-I– and MHC-II–restricted CD8+ T cell responses occurred only in the absence of the Rh157.4–.6/UL128–131 genes involved in CMV tropism for nonfibroblasts. Discussion These findings suggest that CD8+ T cell recognition is more flexible than had been thought, and that the focused epitope recognition profiles of conventional CD8+ T cell responses may be primarily restricted by immunoregulation during priming (which can be subverted by CMV) rather than by intrinsic limitations in antigen processing/presentation or in T cell receptor repertoire. The ability of CMVs with different genetic modifications to differentially elicit CD8+ T cell responses with divergent patterns of epitope recognition raises the possibility of a CMV vector–based vaccine platform with programmable CD8+ T cell epitope targeting, including vectors that can selectively elicit CD8+ T cell responses targeting conventional or unconventional epitopes. Because the latter would be unaffected by escape mutations arising during natural infection, these vectors would be well suited for therapeutic vaccine applications. CMV Breaks All the Rules One vaccine strategy being pursued against HIV is to generate protection that is dependent on cell-mediated, rather than humoral, immune responses. A cytomegalovirus (CMV)–vectored vaccine that expresses simian immunodeficiency virus (SIV) antigens exhibits stringent and durable viral control upon SIV challenge in approximately half of vaccinated rhesus macaques. Hansen et al. (10.1126/science.1237874, see the Perspective by Goonetilleke and McMichael) sought to determine the basis for the protection and discovered that the CD8+ T cell response in vaccinated monkeys does not target canonical SIV epitopes, which SIV is known to escape, but rather generates a broad, promiscuous response. A vaccine that uses one virus to deliver components of a second virus elicits T cells that recognize noncanonical epitopes. [Also see Perspective by Goonetilleke and McMichael] CD8+ T cell responses focus on a small fraction of pathogen- or vaccine-encoded peptides, and for some pathogens, these restricted recognition hierarchies limit the effectiveness of antipathogen immunity. We found that simian immunodeficiency virus (SIV) protein–expressing rhesus cytomegalovirus (RhCMV) vectors elicit SIV-specific CD8+ T cells that recognize unusual, diverse, and highly promiscuous epitopes, including dominant responses to epitopes restricted by class II major histocompatibility complex (MHC) molecules. Induction of canonical SIV epitope–specific CD8+ T cell responses is suppressed by the RhCMV-encoded Rh189 gene (corresponding to human CMV US11), and the promiscuous MHC class I– and class II–restricted CD8+ T cell responses occur only in the absence of the Rh157.5, Rh157.4, and Rh157.6 (human CMV UL128, UL130, and UL131) genes. Thus, CMV vectors can be genetically programmed to achieve distinct patterns of CD8+ T cell epitope recognition.

Progressive CD4+ central–memory T cell decline results in CD4+ effector–memory insufficiency and overt disease in chronic SIV infection

Primary simian immunodeficiency virus (SIV) infections of rhesus macaques result in the dramatic depletion of CD4+ CCR5+ effector–memory T (TEM) cells from extra-lymphoid effector sites, but in most infections, an increased rate of CD4+ memory T cell proliferation appears to prevent collapse of effector site CD4+ TEM cell populations and acute-phase AIDS. Eventually, persistent SIV replication results in chronic-phase AIDS, but the responsible mechanisms remain controversial. Here, we demonstrate that in the chronic phase of progressive SIV infection, effector site CD4+ TEM cell populations manifest a slow, continuous decline, and that the degree of this depletion remains a highly significant correlate of late-onset AIDS. We further show that due to persistent immune activation, effector site CD4+ TEM cells are predominantly short-lived, and that their homeostasis is strikingly dependent on the production of new CD4+ TEM cells from central–memory T (TCM) cell precursors. The instability of effector site CD4+ TEM cell populations over time was not explained by increasing destruction of these cells, but rather was attributable to progressive reduction in their production, secondary to decreasing numbers of CCR5− CD4+ TCM cells. These data suggest that although CD4+ TEM cell depletion is a proximate mechanism of immunodeficiency, the tempo of this depletion and the timing of disease onset are largely determined by destruction, failing production, and gradual decline of CD4+ TCM cells.

Evasion of CD8+ T Cells Is Critical for Superinfection by Cytomegalovirus

Cytomegalovirus Immune Evasion Strategy Cytomegalovirus (CMV) infects a large percentage of the worlds population. Most of those infected are asymptomatic; however, CMV is a substantial public health concern for immunocompromised individuals and neonates. CMV is unusual in that it can superinfect: it re-infects hosts who are already infected with the virus, even in the presence of a strong, specific immune response. Hansen et al. (p. 102; see the Perspective by Hengel and Koszinowski) now find that in rhesus macaques, a good model for human CMV superinfection, CMV establishes superinfections by evading the immune response mediated by CD8+ T cells. A series of viral mutants deficient in expression of the US2-11 glycoproteins, which regulate antigen presentation to CD8+ T cells, revealed that, although able to establish the initial infection, these viral mutants were unable to superinfect. Depletion of CD8+ T cells from the monkeys allowed infection by the mutant viruses. These results highlight the difficulties in developing an effective protective vaccine against CMV itself, but suggest that CMV-based vectors may be useful in other vaccine efforts such as those against HIV. Cytomegalovirus monkeys can reinfect an already-infected host by evading the CD8+ T cell–mediated immune response. Cytomegalovirus (CMV) can superinfect persistently infected hosts despite CMV-specific humoral and cellular immunity; however, how it does so remains undefined. We have demonstrated that superinfection of rhesus CMV–infected rhesus macaques (RM) requires evasion of CD8+ T cell immunity by virally encoded inhibitors of major histocompatibility complex class I (MHC-I) antigen presentation, particularly the homologs of human CMV US2, 3, 6, and 11. In contrast, MHC-I interference was dispensable for primary infection of RM, or for the establishment of a persistent secondary infection in CMV-infected RM transiently depleted of CD8+ lymphocytes. These findings demonstrate that US2-11 glycoproteins promote evasion of CD8+ T cells in vivo, thus supporting viral replication and dissemination during superinfection, a process that complicates the development of preventive CMV vaccines but that can be exploited for CMV-based vector development.

Ureaplasma parvum or Mycoplasma hominis as Sole Pathogens Cause Chorioamnionitis, Preterm Delivery, and Fetal Pneumonia in Rhesus Macaques

The authors assess causal, cellular and inflammatory links between intraamniotic infection with Ureaplasma parvum or Mycoplasma hominis and preterm labor in a nonhuman primate model. Long-term catheterized rhesus monkeys received intraamniotic inoculations of clinical isolates of Ureaplasma parvum serovar 1, M hominis, media control or physiological saline. Genital mycoplasmas were quantified in amniotic fluid (AF) and documented in fetal tissues by culture and PCR. In association with elevated AF colony counts for U parvum or M hominis, there was a sequential upregulation of AF leukocytes, proinflammatory cytokines, prostaglandin E2 and F2a, metalloproteinase-9 and uterine activity ( P< .05). Fetal membranes and lung were uniformly positive for both microorganisms; fetal blood and cerebrospinal fluid cultures and PCR were more often positive for M hominis than U parvum. Histopathologic findings of chorioamnionitis, a systemic fetal inflammatory response and pneumonitis worsen with duration of in utero infection. U parvum or M hominis, as sole pathogens, elicit a robust proinflammatory response which contributes to preterm labor and fetal lung injury.

Lymph node T cell responses predict the efficacy of live attenuated SIV vaccines.

Live attenuated simian immunodeficiency virus (SIV) vaccines (LAVs) remain the most efficacious of all vaccines in nonhuman primate models of HIV and AIDS, yet the basis of their robust protection remains poorly understood. Here we show that the degree of LAV-mediated protection against intravenous wild-type SIVmac239 challenge strongly correlates with the magnitude and function of SIV-specific, effector-differentiated T cells in the lymph node but not with the responses of such T cells in the blood or with other cellular, humoral and innate immune parameters. We found that maintenance of protective T cell responses is associated with persistent LAV replication in the lymph node, which occurs almost exclusively in follicular helper T cells. Thus, effective LAVs maintain lymphoid tissue-based, effector-differentiated, SIV-specific T cells that intercept and suppress early wild-type SIV amplification and, if present in sufficient frequencies, can completely control and perhaps clear infection, an observation that provides a rationale for the development of safe, persistent vectors that can elicit and maintain such responses.