Scott G. Hansen

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.

Duration of antiviral immunity after smallpox vaccination

Although naturally occurring smallpox was eliminated through the efforts of the World Health Organization Global Eradication Program, it remains possible that smallpox could be intentionally released. Here we examine the magnitude and duration of antiviral immunity induced by one or more smallpox vaccinations. We found that more than 90% of volunteers vaccinated 25–75 years ago still maintain substantial humoral or cellular immunity (or both) against vaccinia, the virus used to vaccinate against smallpox. Antiviral antibody responses remained stable between 1–75 years after vaccination, whereas antiviral T-cell responses declined slowly, with a half-life of 8–15 years. If these levels of immunity are considered to be at least partially protective, then the morbidity and mortality associated with an intentional smallpox outbreak would be substantially reduced because of pre-existing immunity in a large number of previously vaccinated individuals.

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.

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.

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.

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.

Complete Sequence and Genomic Analysis of Rhesus Cytomegalovirus

ABSTRACT The complete DNA sequence of rhesus cytomegalovirus (RhCMV) strain 68-1 was determined with the whole-genome shotgun approach on virion DNA. The RhCMV genome is 221,459 bp in length and possesses a 49% G+C base composition. The genome contains 230 potential open reading frames (ORFs) of 100 or more codons that are arranged colinearly with counterparts of previously sequenced betaherpesviruses such as human cytomegalovirus (HCMV). Of the 230 RhCMV ORFs, 138 (60%) are homologous to known HCMV proteins. The conserved ORFs include the structural, replicative, and transcriptional regulatory proteins, immune evasion elements, G protein-coupled receptors, and immunoglobulin homologues. Interestingly, the RhCMV genome also contains sequences with homology to cyclooxygenase-2, an enzyme associated with inflammatory processes. Closer examination identified a series of candidate exons with the capacity to encode a full-length cyclooxygenase-2 protein. Counterparts of cyclooxygenase-2 have not been found in other sequenced herpesviruses. The availability of the complete RhCMV sequence along with the ability to grow RhCMV in vitro will facilitate the construction of recombinant viral strains for identifying viral determinants of CMV pathogenicity in the experimentally infected rhesus macaque and to the development of CMV as a vaccine vector.

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.

New paradigms for HIV/AIDS vaccine development.

HIV-1 and its simian counterpart SIV have been exquisitely tailored by evolution to evade host immunity. By virtue of specific adaptations that thwart individual innate or adaptive immune mechanisms, and an overall replication strategy that provides for rapid establishment of a large, systemic viral population, capable of dynamic adaptation to almost all immune selection pressures, these viruses, once established, almost invariably stay one step ahead of the hosts immune system, and in the vast majority of infected individuals, replicate indefinitely. Although many vaccine approaches tested to date have been able to enhance the magnitude of the immune responses to HIV/SIV infection, most of these responses, whether cellular or humoral, have largely failed to be both effectively antiviral and targeted to prevent the emergence of fully functional escape variants. Recent advances, however, have provided strong evidence that the initial stages of infection following mucosal transmission of these viruses are more vulnerable to immune intervention, and have led to the development of vaccine strategies that elicit responses able to effectively intervene in these early stages of infection, either preventing acquisition of infection or establishing early, stringent, and durable control. Here, we place HIV/AIDS vaccine development in the context of the basic immunobiology of HIV and SIV, review the evidence for their vulnerability to immune responses immediately after mucosal transmission, and discuss how this newly recognized vulnerability might be exploited for the development of an effective HIV/AIDS vaccine.

Broadly targeted CD8+ T cell responses restricted by major histocompatibility complex-E

An unconventional route to protection One promising approach toward an HIV-1 vaccine involves infecting people with cytomegalovirus engineered to express proteins from HIV-1. This approach, which works by eliciting virus-killing CD8+ T cells, provides robust protection in nonhuman primate models. Hansen et al. have found out why this approach is so effective. Normally, peptide antigens presented by major histocompatibility complex-1a (MHC-Ia) activate CD8+ T cells. In vaccinated monkeys, however, CD8+ T cells reacted to peptide antigens presented by MHC-E molecules instead. Moreover, MHC-E could present a much wider range of peptides than MHC-Ia. Science, this issue p. 714 Nonclassical major histocompatibility complex E molecules can present highly diverse peptide epitopes to CD8+ T cells. Major histocompatibility complex E (MHC-E) is a highly conserved, ubiquitously expressed, nonclassical MHC class Ib molecule with limited polymorphism that is primarily involved in the regulation of natural killer (NK) cells. We found that vaccinating rhesus macaques with rhesus cytomegalovirus vectors in which genes Rh157.5 and Rh157.4 are deleted results in MHC-E–restricted presentation of highly varied peptide epitopes to CD8αβ+ T cells, at ~4 distinct epitopes per 100 amino acids in all tested antigens. Computational structural analysis revealed that MHC-E provides heterogeneous chemical environments for diverse side-chain interactions within a stable, open binding groove. Because MHC-E is up-regulated to evade NK cell activity in cells infected with HIV, simian immunodeficiency virus, and other persistent viruses, MHC-E–restricted CD8+ T cell responses have the potential to exploit pathogen immune-evasion adaptations, a capability that might endow these unconventional responses with superior efficacy.