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Despite widespread use of the bacille Calmette-Guérin (BCG) vaccine, tuberculosis (TB) remains a leading cause of global mortality from a single infectious agent (Mycobacterium tuberculosis or Mtb). Here, over two independent Mtb challenge studies, we demonstrate that subcutaneous vaccination of rhesus macaques (RMs) with rhesus cytomegalovirus vectors encoding Mtb antigen inserts (hereafter referred to as RhCMV/TB)--which elicit and maintain highly effector-differentiated, circulating and tissue-resident Mtb-specific CD4[sup.+] and CD8[sup.+] memory T cell responses--can reduce the overall (pulmonary and extrapulmonary) extent of Mtb infection and disease by 68%, as compared to that in unvaccinated controls, after intrabronchial challenge with the Erdman strain of Mtb at [similar]1 year after the first vaccination. Fourteen of 34 RhCMV/TB-vaccinated RMs (41%) across both studies showed no TB disease by computed tomography scans or at necropsy after challenge (as compared to 0 of 17 unvaccinated controls), and ten of these RMs were Mtb-culture-negative for all tissues, an exceptional long-term vaccine effect in the RM challenge model with the Erdman strain of Mtb. These results suggest that complete vaccine-mediated immune control of highly pathogenic Mtb is possible if immune effector responses can intercept Mtb infection at its earliest stages., Author(s): Scott G Hansen [1]; Daniel E Zak [2]; Guangwu Xu [1]; Julia C Ford [1]; Emily E Marshall [1]; Daniel Malouli [1]; Roxanne M Gilbride [1]; Colette M Hughes [...]

Cellular immune responses in rhesus macaques (Macaca mulatta) vaccinated with cytomegalovirus vectors expressing SIV proteins are able to stringently control highly pathogenic SIV infection, regardless of the route of challenge, after systemic spread; immunological and virological analyses of protected macaques followed for up to 3 years suggest that persistent immune surveillance by vaccine-elicited immune responses may have cleared the infection. Vaccine combats virulent SIV infection Work on potential vaccines against human immunodeficiency viruses (HIV) and the simian equivalent (SIV) has so far proved largely fruitless. This study makes some progress by exploiting the recent observation that the pathogens appear vulnerable to immune control or pharmacological clearance in the first few hours to days of infection. Rhesus macaques vaccinated with SIV protein-expressing rhesus cytomegalovirus (RhCMV/SIV) vectors developed durable resistance to the highly pathogenic SIVmac239 after challenge by vaginal and intravenous routes. Some of the vaccinated animals have controlled viral replication for 1 to 3 years with no demonstrable evidence for residual virus, raising the possibility that the vaccine- elicited immune responses may in fact have cleared the initial 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.sup.1,2,3,4. 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., Author(s): Scott G. Hansen [sup.1] , Michael Piatak Jr [sup.2] , Abigail B. Ventura [sup.1] , Colette M. Hughes [sup.1] , Roxanne M. Gilbride [sup.1] , Julia C. Ford [sup.1] [...]

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., It has been two decades since the first report that LAV administration can completely protect rhesus macaques from subsequent challenge with highly pathogenic, wild-type SIV(1), a degree of efficacy that [...]

The acquired immunodeficiency syndrome (AIDS)-causing lenti-viruses 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 (1-3). 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 (4). Here we report that SIV vaccines that include rhesus cytomegalovirus (RhCMV) vectors (5) establish indefinitely persistent, high-frequency, SIV-specific effector memory T-cell ([T.sub.EM]) responses at potential sites of SIV replication in rhesus macaques and stringently control highly pathogenic [SIV.sub.MAC239] 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.sup.+] 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.sup.+] 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.sup.+] or [CD4.sup.+] 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 [T.sub.EM] responses might significantly contribute to an efficacious HIV/AIDS vaccine., Conventional prime-boost vaccine regimens with non-persistent vectors lead to lymphoid tissue-based memory T-cell responses ('central memory' or [T.sub.EM]), which deliver peak effector responses only after [T.sub.CM] cells have undergone antigen-stimulated [...]

Author(s): Scott G. Hansen; Michael Piatak; Abigail B. Ventura; Colette M. Hughes; Roxanne M. Gilbride; Julia C. Ford; Kelli Oswald; Rebecca Shoemaker; Yuan Li; Matthew S. Lewis; Awbrey N. Gilliam; [...]

Approximately 50% of the US population received smallpox vaccinations before routine immunization ceased in 1972 for civilians and in 1990 for military personnel. Several studies have shown long-term immunity after smallpox vaccination, but skepticism remains as to whether this will translate into full protection against the onset of orthopoxvirus-induced disease. The US monkeypox outbreak of 2003 provided the opportunity to examine this issue. Using independent and internally validated diagnostic approaches with [greater than or equal to]95% sensitivity and [greater than or equal to]90% specificity for detecting clinical monkeypox infection, we identified three previously unreported cases of monkeypox in preimmune individuals at 13, 29 and 48 years after smallpox vaccination. These individuals were unaware that they had been infected because they were spared any recognizable disease symptoms. Together, this shows that the US monkeypox outbreak was larger than previously realized and, more importantly, shows that cross-protective antiviral immunity against West African monkeypox can potentially be maintained for decades after smallpox vaccination., Author(s): Erika Hammarlund [1]; Matthew W Lewis [1]; Shirley V Carter [1]; Ian Amanna [1]; Scott G Hansen [1]; Lisa I Strelow [1]; Scott W Wong [1]; Paul Yoshihara [1]; [...]

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., Author(s): Erika Hammarlund [1]; Matthew W Lewis [1]; Scott G Hansen [1]; Lisa I Strelow [1]; Jay A Nelson [1]; Gary J Sexton [2]; Jon M Hanifin [3]; Mark K [...]

Prophylactic vaccination of rhesus macaques with rhesus cytomegalovirus (RhCMV) vectors expressing simian immunodeficiency virus (SIV) antigens (RhCMV/SIV) elicits immune responses that stringently control highly pathogenic SIV infection, with subsequent apparent clearance of the infection, in ~50% of vaccinees. In contrast, here, we show that therapeutic RhCMV/SIV vaccination of rhesus macaques previously infected with SIV and given continuous combination antiretroviral therapy (cART) beginning 4-9 d post-SIV infection does not mediate measurable SIV reservoir clearance during over 600 d of follow-up on cART relative to RhCMV/control vaccination. However, none of the six animals started on cART on day four or five, across both RhCMV/SIV- and RhCMV/control-vaccinated groups, those rhesus macaques with SIV reservoirs most closely resembling those of prophylactically RhCMV/SIV-vaccinated and protected animals early in their course, showed post-cART viral rebound with up to nine months of follow-up. Moreover, at necropsy, these rhesus macaques showed little to no evidence of replication-competent SIV. These results suggest that the early SIV reservoir is limited in durability and that effective blockade of viral replication and spread in this critical time window by either pharmacologic or immunologic suppression may result in reduction, and potentially loss, of rebound-competent virus over a period of ~two years. Early and prolonged administration of antiretroviral therapy to SIV-infected and post-exposure-vaccinated rhesus macaques was associated with absence or delay of detectable virus after therapy interruption., Author(s): Afam A. Okoye [sup.1] , Scott G. Hansen [sup.1] , Mukta Vaidya [sup.1] , Yoshinori Fukazawa [sup.1] , Haesun Park [sup.1] , Derick M. Duell [sup.1] , Richard Lum [...]

Nature 502, 100-104 (2013); doi:10.1038/nature12519 The Acknowledgements section of this Letter should have included the following sentence: 'We acknowledge the contribution of M. A. Jarvis to the design, construction and initial in vitro characterization of all the strain 68.1-derived RhCMV vectorsused in this study, including both previously published RhCMV/SIV vectors and a previously unpublished vector expressing an Mycobacterium tuberculosis Ag85B-ESAT6 fusion protein, used as a negative control in this study., Author(s): Scott G. Hansen, Michael Piatak, Abigail B. Ventura, Colette M. Hughes, Roxanne M. Gilbride, Julia C. Ford, Kelli Oswald, Rebecca Shoemaker, Yuan Li, Matthew S. Lewis, Awbrey N. Gilliam, [...]

In Supplementary Figure 1 of our paper, the reference cited (Hel et al. in Vaccine 20, 3171-3186, 2002) and the description for the Retanef fusion gene expressed by RhCMV-Retanef were [...]

Vaccines based on cytomegalovirus (CMV) demonstrate protection in animal models of infectious disease and cancer. Vaccine efficacy is associated with the ability of CMV to elicit and indefinitely maintain high frequencies of circulating effector memory T cells (T EM ) providing continuous, life-long anti-pathogen immune activity. To allow for the clinical testing of human CMV (HCMV)-based vaccines we constructed and characterized as a vector backbone the recombinant molecular clone TR3 representing a wildtype genome. We demonstrate that TR3 can be stably propagated in vitro and that, despite species incompatibility, recombinant TR3 vectors elicit high frequencies of T EM to inserted antigens in rhesus macaques (RM). Live-attenuated versions of TR3 were generated by deleting viral genes required to counteract intrinsic and innate immune responses. In addition, we eliminated subunits of a viral pentameric glycoprotein complex thus limiting cell tropism. We show in a humanized mouse model that such modified vectors were able to establish persistent infection but lost their ability to reactivate from latency. Nevertheless, attenuated TR3 vectors preserved the ability to elicit and maintain T EM to inserted antigens in RM. We further demonstrate that attenuated TR3 can be grown in approved cell lines upon elimination of an anti-viral host factor using small interfering RNA, thus obviating the need for a complementing cell line. In sum, we have established a versatile platform for the clinical development of live attenuated HCMV-vectored vaccines and immunotherapies.