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Numerous attempts to produce antiviral vaccines by harnessing memory CD8 T cells have failed. A barrier to progress is that we do not know what makes an Ag a viable target of protective CD8 T cell memory. We found that in mice susceptible to lethal mousepox (the mouse homolog of human smallpox), a dendritic cell vaccine that induced memory CD8 T cells fully protected mice when the infecting virus produced Ag in large quantities and with rapid kinetics. Protection did not occur when the Ag was produced in low amounts, even with rapid kinetics, and protection was only partial when the Ag was produced in large quantities but with slow kinetics. Hence, the amount and timing of Ag expression appear to be key determinants of memory CD8 T cell antiviral protective immunity. These findings may have important implications for vaccine design.

Nuclear factor kappa B (NF-κB) and type 1 interferon (T1-IFN) signaling are innate immune mechanisms activated upon viral infection. However, the role of NF-κB and its interplay with T1-IFN in antiviral immunity is poorly understood. We show that NF-κB is essential for resistance to ectromelia virus (ECTV), a mouse orthopoxvirus related to the virus causing human smallpox. Additionally, an ECTV mutant lacking an NF-κB inhibitor activates NF-κB more effectively in vivo, resulting in increased proinflammatory molecule transcription in uninfected cells and organs and decreased viral replication. Unexpectedly, NF-κB activation compensates for genetic defects in the T1-IFN pathway, such as a deficiency in the IRF7 transcription factor, resulting in virus control. Thus, overlap between the T1-IFN and NF-κB pathways allows the host to overcome genetic or pathogen-induced deficiencies in T1-IFN and survive an otherwise lethal poxvirus infection. These findings may also explain why some pathogens target both pathways to cause disease. © 2013 Elsevier Inc., NIAID (U19AI083008 and R01AI065544); NCI (CA006927); Instituto de Salud Carlos III, Spanish Ministry of Health; Spanish Ministry of Science and Innovation

The two major antiviral effector mechanisms of CD8 + T cells are thought to be perforin (Prf)-mediated cell lysis and gamma interferon (IFN-γ)-mediated induction of an antiviral state. By affecting the expression of proteins involved in antigen presentation, IFN-γ is also thought to shape the magnitude and specificity of the CD8 + T cell response. Here we studied the roles of Prf and IFN-γ in shaping the effector and memory CD8 + T cell responses to vaccinia virus (VACV). IFN-γ deficiency resulted in increased numbers of anti-VACV effector and memory CD8 + T cells, which were partly dependent on increased virus loads. On the other hand, Prf-deficient mice showed an increase in the number of VACV-specific CD8 + T cells only in the memory phase. Treatment of the mice with the antiviral drug cidofovir reduced the numbers of effector and memory cells closer to wild-type levels in IFN-γ-deficient mice and reduced the numbers of memory CD8 + T cells to wild-type levels in Prf-deficient mice. These data suggest that virus loads are the main reason for the increased strength of the CD8 response in IFN-γ- and Prf-deficient mice. Neither Prf deficiency nor IFN-γ deficiency had an effect on the immunodominance hierarchy of five K b -restricted CD8 + T cell determinants either during acute infection or after recovery. Thus, our work shows that CD8 + T cell immunodominance during VACV infection is not affected by the effects of IFN-γ on the antigen presentation machinery.

SummaryToll-like receptor 9 (TLR9), its adaptor MyD88, the downstream transcription factor interferon regulatory factor 7 (IRF7), and type I interferons (IFN-I) are all required for resistance to infection with ectromelia virus (ECTV). However, it is not known how or in which cells these effectors function to promote survival. Here, we showed that after infection with ECTV, the TLR9-MyD88-IRF7 pathway was necessary in CD11c+ cells for the expression of proinflammatory cytokines and the recruitment of inflammatory monocytes (iMos) to the draining lymph node (dLN). In the dLN, the major producers of IFN-I were infected iMos, which used the DNA sensor-adaptor STING to activate IRF7 and nuclear factor κB (NF-κB) signaling to induce the expression of IFN-α and IFN-β, respectively. Thus, in vivo, two pathways of DNA pathogen sensing act sequentially in two distinct cell types to orchestrate resistance to a viral disease.

It has been shown in various infection models that CD4 + T cell help (T H ) is necessary for the conditioning, maintenance, and/or recall responses of memory CD8 + T cells (CD8 M) . Yet, in the case of vaccinia virus (VACV), which constitutes the vaccine used to eradicate smallpox and is a candidate vector for other infectious diseases, the issue is controversial because different groups have shown either T H dependence or independence of CD8 M conditioning, maintenance, and/or recall response. In agreement with some of these groups, we show that T H plays a role in, but is not essential for, the maintenance, proliferation, and effector differentiation of polyclonal memory CD8 + T cells after infection with wild-type VACV strain Western Reserve. More important, we show that unhelped and helped anti-VACV memory CD8 + T cells are similarly efficient at protecting susceptible mice from lethal mousepox, the mouse equivalent of human smallpox. Thus, T H is not essential for the conditioning and maintenance of memory CD8 + T cells capable of mounting a recall response strong enough to protect from a lethal natural pathogen. Our results may partly explain why the VACV vaccine is so effective. IMPORTANCE We used vaccinia virus (VACV)—a gold standard vaccine—as the immunogen and ectromelia virus (ECTV) as the pathogen to demonstrate that the conditioning and maintenance of anti-VACV memory CD8 + T cells and their ability to protect against an orthopoxvirus (OPV) infection in its natural host can develop in the absence of CD4 + T cell help. Our results provide important insight to our basic knowledge of the immune system. Further, because VACV is used as a vaccine in humans, our results may help us understand how this vaccine induces protective immunity in this species. In addition, this work may partly explain why VACV is so effective as a vaccine.

Immunization with vaccinia virus (VACV), the virus comprising the smallpox vaccine, induces memory CD8+ T cells that protect from subsequent infections with smallpox in humans or the related ectromelia virus (ECTV) in mice. Memory CD8+ T cells largely mediate these effects by expanding into secondary effectors that secrete the antiviral cytokine interferon-γ (IFN-γ) and induce cytolysis via releasing factors such as perforin, which permeabilizes target cells. We show that protection from ECTV infection after VACV immunization depends on the initial memory cell frequency and ability of expanded secondary effectors to kill infected targets in a perforin-dependent manner. Although IFN-γ is essential for antiviral protection, it can be produced by either secondary effectors or concomitant primary effector CD8 + T cells recruited to the response. Thus, during lethal virus challenge, memory CD8+ T cells are required for cytolytic killing of infected cells, but primary effectors can play important roles by producing IFN-γ. © 2013 Elsevier Inc., R01AI048849, R01AI065544, 5U19AI083008; P30CA006927; NIH T32 CA-009035036

Memory CD8⁺ T cells play an essential role in controlling pathogenic infections. Therefore generating protective memory CD8⁺ T cells by vaccination is an attractive strategy for preventing and treating a variety of human diseases. Understanding what comprises a protective memory CD8⁺ T cell response will help optimize vaccine-induced CD8⁺ T cell immunity. Here we discuss essential antiviral effector functions and highlight how recall expansion of memory CD8⁺ T cells may affect the primary response.

Memory CD8+ T cells play an essential role in controlling pathogenic infections. Therefore generating protective memory CD8+ T cells by vaccination is an attractive strategy for preventing and treating a variety of human diseases. Understanding what comprises a protective memory CD8+ T cell response will help optimize vaccine-induced CD8+ T cell immunity. Here we discuss essential antiviral effector functions and highlight how recall expansion of memory CD8+ T cells may affect the primary response.

The antigens recognized by individual CD8 + T cells are small peptides bound to major histocompatibility complex (MHC) class I molecules. The CD8 + T cell response to a virus is restricted to several peptides, and the magnitudes of the effector as well as memory phases of the response to the individual peptides are generally hierarchical. The peptide eliciting a stronger response is called immunodominant (ID), and those with smaller-magnitude responses are termed subdominant (SD). The relative importance of ID and SD determinants in protective immunity remains to be fully elucidated. We previously showed that multispecific memory CD8 + T cells can protect susceptible mice from mousepox, an acute lethal viral disease. It remained unknown, however, whether CD8 + T cells specific for single ID or SD peptides could be protective. Here, we demonstrate that immunization with dendritic cells pulsed with ID and some but not all SD peptides induces memory CD8 + T cells that are fully capable of protecting susceptible mice from mousepox. Additionally, while natural killer (NK) cells are essential for the natural resistance of nonimmune C57BL/6 (B6) to mousepox, we show that memory CD8 + T cells of single specificity also protect B6 mice depleted of NK cells. This suggests it is feasible to produce effective antiviral CD8 + T cell vaccines using single CD8 + T cell determinants and that NK cells are no longer essential when memory CD8 + T cells are present. © 2012, American Society for Microbiology.

The extent to which direct- and cross-presentation (DP and CP) contribute to the priming of CD8+ T cell (TCD8+) responses to viruses is unclear mainly because of the difficulty in separating the two processes. Hence, while CP in the absence of DP has been clearly demonstrated, induction of an anti-viral TCD8+ response that excludes CP has never been purposely shown. Using vaccinia virus (VACV), which has been used as the vaccine to rid the world of smallpox and is proposed as a vector for many other vaccines, we show that DP is the main mechanism for the priming of an anti-viral TCD8+ response. These findings provide important insights to our understanding of how one of the most effective anti-viral vaccines induces immunity and should contribute to the development of novel vaccines., Author Summary Professional antigen presenting cells fragment viral proteins and display some of the resulting peptides bound to MHC molecules at the cell surface. When virus-specific CD8+ T cells recognize these viral peptides they become activated, proliferate, and kill virus-infected cells to help rid the body of the virus. Two pathways have been described for the origin of the peptides presented by professional antigen presenting cells. In cross-presentation, the antigen presenting cells acquire the proteins from other cells which, in the case of a viral infection, must be infected. In direct presentation, the antigen presenting cells synthesize the proteins themselves and, therefore, during responses to viruses must be infected. However, the participation of direct presentation in anti-viral responses has never been deliberately demonstrated experimentally. In this paper we demonstrate that direct presentation occurs and is the main pathway to induce CD8+ T cells during infection with vaccinia virus. These findings provide important insights to our understanding of how one of the most effective anti-viral vaccines induces immunity and should contribute to the development of novel vaccines.

Ectromelia virus (ECTV), the causative agent of mousepox, is a lethal mouse pathogen of the Orthopoxvirus genus, which also includes variola virus. C57Bl/6 (B6) mice are resistant to ECTV infection while mice such as DBA2/J are susceptible. B6.D2-D6 mice, a B6 congenic strain with the NK complex from DBA2/J are sensitive to ECTV and can be protected by adoptive transfer of memory CD8+ T cells (MTCD8+) from convalescent mice. To ascertain the role of IFNγ in MTCD8+ mediated protection, we transferred MTCD8+ from IFNγ-/- mice (Mγ-/-) into B6.D2-D6 mice and challenged them with ECTV. All naïve CD8+ (NB6) recipients died and all recipients of memory B6 CD8+ (MB6) or Mγ-/- recipients survived. Seven dpi, MB6 and Mγ-/- recipients had significantly less liver damage and virus titers in the spleen and liver than NB6 recipients. This demonstrates that MTCD8+ do not have to produce IFNγ to protect from a viral disease. Of interest, we also found that donor virus-specific cells promoted an endogenous virus-specific TCD8+ response in the spleens and livers of recipients. Next we determined if IFNγ was necessary when MTCD8+ cells were present by transferring MB6 or Mγ-/- CD8+ into IFNγ-/- mice. All NB6 and Mγ-/- recipients succumbed to mousepox while all recipients of MB6 recipients survived. Thus, IFNγ is required for resistance to mousepox but can be produced in sufficient quantities for protection by MTCD8+ or other non-memory cells, probably NK cells or primary T cell effectors.