A novel approach to visualize polyclonal virus-specific CD8 T cells in vivo

Recent technical breakthroughs in generating soluble MHC class I-peptide tetramers now allow the direct visualization of virus-specific CD8 T cells after infection in vivo. However, this technique requires the knowledge of the immunodominant viral epitopes recognized by T cells. Here, we describe an alternative approach to visualize polyclonal virus-specific CD8 T cells in vivo using a simple adoptive transfer system. In our approach, C57BL/6 (Thy1.2) mice were infected with lymphocytic choriomeningitis virus, vesicular stomatitis virus, or vaccinia virus to induce virus-specific memory T cells. Tracer T cells (2 x 106) from these virus-immune mice were adoptively transferred into nonirradiated (C57BL/6 x B6.PL-Thy-1a)F1 mice. After infection of the F1-recipient mice with the appropriate virus, the transferred cells expanded vigorously, and on day 8 postinfection 60-80% of total CD8 T cells were of donor T cell origin. Under the same conditions memory CD4 T cells gave rise to at least 10 times less cell numbers than memory CD8 T cells. The transfer system described here not only allows to visualize effector and memory CD8 T cells in vivo but also to isolate them for further in vitro characterization without knowing the epitopes recognized by these Ag-specific CD8 T cells.     

Memories of virus-specific CD8+ T cells

This brief review focuses on the way that our understanding of virus-specific CD8(+) T-cell-mediated immunity evolved, giving particular attention to the early impact of the program at the Australian National University. The story developed through a sequence of distinct eras, each of which can be defined in the context of the technologies available at that time. The progress has been enormous, but there is a great deal still to be learned. A particular challenge is to use what we know for human benefit.     

Protective and pathological roles of virus-specific and bystander CD8+ T cells in herpetic stromal keratitis

Herpetic stromal keratitis (HSK), resulting from corneal HSV-1 infection, represents a T cell-mediated immunopathologic lesion. In T cell transgenic mice on a SCID or RAG knockout background, the T cells mediating lesions are unreactive to viral Ags. In these bystander models, animals develop ocular lesions but are unable to control infection. Transfer of HSV-immune cells into a CD8(+) T cell bystander model resulted in clearance of virus from eyes, animals survived, and lesions developed to greater severity. However, the adoptively transferred CD8(+) T cells were not evident in lesions, although they were readily detectable in the lymphoid tissues as well as in the peripheral and CNS. Our results indicate that viral-induced tissue damage can be caused by bystander cells, but these fail to control infection. Immune CD8(+) T cells trigger clearance of virus from the eye, but this appears to result by the T cells acting at sites distal to the cornea. A case is made that CD8(+) T cell control is expressed in the trigeminal ganglion, serving to curtail a source of virus to the cornea.     

Enhanced expression of cell cycle regulatory genes in virus-specific memory CD8+ T cells

Unlike naive CD8+ T cells, antigen-experienced memory CD8+ T cells persist over time due to their unique ability to homeostatically proliferate. It was hypothesized that memory cells might differentially regulate the expression of genes that control the cell cycle to facilitate homeostatic proliferation. To test this, the expression levels of 96 different cell cycle regulatory genes were compared between transgenic naive and memory CD8+ T cells that specifically recognize the GP33-41 epitope of lymphocytic choriomeningitis virus (LCMV). It was discovered that relative to naive cells, memory cells overexpress several important genes that control the transition between G(1) and S phase. Some of these genes include those encoding cyclins D3, D2, B1, C, and H, cyclin-dependent kinases (cdk's) 4 and 6, the cdk inhibitors p16, p15, and p18, and other genes involved in protein degradation and DNA replication. Importantly, these differences were observed both in total populations of LCMV-specific naive and memory CD8+ cells and in LCMV-specific CD8+ T-cell populations that were in the G(1) phase of the cell cycle only. In addition, the expression differences between naive and memory cells were exaggerated following antigenic stimulation. The fact that memory cells are precharged with several of the major factors that are necessary for the G(1)- to-S-phase transition suggests they may require a lower threshold of stimulation to enter the cell cycle.     

Dendritic cells and CD28 costimulation are required to sustain virus-specific CD8+ T cell responses during the effector phase in vivo

Although much is known about the initiation of immune responses, much less is known about what controls the effector phase. CD8(+) T cell responses are believed to be programmed in lymph nodes during priming without any further contribution by dendritic cells (DCs) and Ag. In this study, we report the requirement for DCs, Ag, and CD28 costimulation during the effector phase of the CD8(+) T cell response. Depleting DCs or blocking CD28 after day 6 of primary influenza A virus infection decreases the virus-specific CD8(+) T cell response by inducing apoptosis, and this results in decreased viral clearance. Furthermore, effector CD8(+) T cells adoptively transferred during the effector phase fail to expand without DC, CD28 costimulation, and cognate Ag. The absence of costimulation also leads to reduced survival of virus-specific effector cells as they undergo apoptosis mediated by the proapoptotic molecule Bim. Finally, IL-2 treatment restored the effector response in the absence of CD28 costimulation. Thus, in contrast to naive CD8(+) T cells, which undergo an initial Ag-independent proliferation, effector CD8(+) T cells expanding in the lungs during the effector phase require Ag, CD28 costimulation, and DCs for survival and expansion. These requirements would greatly impair effector responses against viruses and tumors that are known to inhibit DC maturation and in chronic infections and aging where CD28(-/-) CD8(+) T cells accumulate.     

Self-reactive T cell receptor-reactive CD8+ T cells inhibit T cell lymphoma growth in vivo

Syngenic C57BL/6 mice (H-2(b)) vaccinated with mitomycin C-treated L12R4 T lymphoma cells develop protective immunity toward the MHC class II-negative tumor cells. In the present study, we characterize the nature, mode of function, and specificity of the effector cells in this immunity. These cells are TCR-specific CD8(+) T lymphocytes with effector function in vitro as well as in vivo upon transfer to naive mice. They produce high levels of IFN-gamma and TNF-alpha, but little or no IL-4. By means of TCRbeta-negative variant L12R4 cells, P3.3, and TCR-Vbeta2 cDNA-transfected and TCR-Vbeta2-expressing P3.3 lymphoma cells, we found that a significant part of the effector T cells are specific for the Vbeta12 region. The growth inhibition of L12R4 cells in vitro was inhibited by anti-H-2, anti-K(b), and anti-D(b) mAb. Furthermore, vaccination with Vbeta12 peptide p67-78, which binds to both K(b) and D(b) MHC class I molecules, induces partial protection against L12R4 T lymphoma cells. Thus, self-reactive TCR-Vbeta-specific, K(b)-, or D(b)-restricted CD8(+) T cells mediate inhibition of T cell lymphoma growth in vitro and in vivo.     

Visualization of polyoma virus-specific CD8+ T cells in vivo during infection and tumor rejection.

T cells are critical for clearing infection and preventing tumors induced by polyoma virus, a natural murine papovavirus. We previously identified the immunodominant epitope for polyoma virus-specific CTL in tumor-resistant H-2k mice as the Dk-restricted peptide, MT389-397, derived from the polyoma middle T oncoprotein. In this study, we developed tetrameric Dk complexes containing the MT389-397 peptide to directly visualize and enumerate MT389-397-specific CTL during polyoma virus infection. We found that Dk/MT389 tetramer+CD8+ T cells undergo a massive expansion during primary infection such that by day 7 postinfection these Ag-specific CD8+ T cells constitute approximately 20% of the total and approximately 40% of the activated CD8+ T cells in the spleen. This expansion of Dk/MT389 tetramer+CD8+ T cells parallels the emergence of MT389-397-specific ex vivo cytolytic activity and clearance of polyoma virus. Notably, Dk/MT389 tetramer+CD8+ T cells are maintained in memory at very high levels. The frequencies of Dk/MT389 tetramer+CD8+ effector and memory T cells in vivo match those of CD8+ T cells producing intracellular IFN-gamma after 6-h in vitro stimulation by MT389-397 peptide. Consistent with preferential Vbeta6 expression by MT389-397-specific CD8+CTL lines and clones, Dk/MT389 tetramer+CD8+ T cells exhibit biased expression of this Vbeta gene segment. Finally, we show that Dk/MT389 tetramer+CD8+ T cells efficiently infiltrate a polyoma tumor challenge to virus-immune mice. Taken together, these findings strongly implicate virus-induced MT389-397-specific CD8+ T cells as essential effectors in eliminating polyoma-infected and polyoma-transformed cells in vivo.     

An in vivo cytotoxicity threshold for influenza A virus-specific effector and memory CD8(+) T cells

Influenza A virus infection of C57BL/6 (B6) mice is characterized by prominent CD8(+) T cell responses to H2D(b) complexed with peptides from the viral nucleoprotein (NP(366), ASNENMETM) and acid polymerase (PA(224), SSLENFRAYV). An in vivo cytotoxicity assay that depends on the adoptive transfer of peptide-pulsed, syngeneic targets was used in this study to quantitate the cytotoxic potential of D(b)NP(366)- and D(b)PA(224)-specific acute and memory CD8(+) T cells following primary or secondary virus challenge. Both T cell populations displayed equivalent levels of in vivo effector function when comparable numbers were transferred into naive B6 hosts. Cytotoxic activity following primary infection clearly correlated with the frequency of tetramer-stained CD8(+) T cells. This relationship looked, however, to be less direct following secondary exposure, partly because the numbers of CD8(+)D(b)NP(366)(+) T cells were greatly in excess. However, calculating the in vivo E:T ratios indicated that in vivo lysis, like many other biological functions, is threshold dependent. Furthermore, the capacity to eliminate peptide-pulsed targets was independent of the differentiation state (i.e., primary or secondary effectors) and was comparable for the two T cell specificities that were analyzed. These experiments provide insights that may be of value for adoptive immunotherapy, where careful consideration of both the activation state and the number of effector cells is required.     

Persistent suppression of virus-specific cytotoxic T cell responses after transient depletion of CD4+ T cells in vivo

Co-administration of soluble Ag and anti-CD4 mAb has been successfully used to induce long term Ag-specific tolerance. The mechanisms underlying persistent immunologic unresponsiveness are unclear. We have now studied whether tolerance toward complex viral Ag expressed on Moloney sarcoma virus (MSV)-transformed tumor cells can be induced when given at the time of severe helper cell depletion. Although mice that had been injected with anti-CD4 mAb at the time of immunization regained the ability to recognize MSV Ag, their humoral and cytotoxic immunity to MSV were severely compromised. Ag-specific low responsiveness was maintained for more than 6 mo. To analyze the T cell repertoire of low responder mice we have estimated precursor frequencies of MSV-specific proliferative and cytotoxic T cells after the CD4+ T cell subset was fully reconstituted. There was no difference in the frequencies of control and low responder mice excluding clonal deletion as the mechanism maintaining low responsiveness. In co-culture experiments the defect in low responder mice could be localized to the regenerated CD4+ T cell subset, suggesting the induction of CD4+ suppressor-inducer cells. Alternatively, regenerated CD4+ cells in anti-CD4 conditioned mice had acquired a defect to provide help for MSV-specific responses. In spite of the potentials to induce low responsiveness to selected Ag by anti-CD4 conditioning, the risk to cause persistent virus-specific immunodeficiency might limit the clinical application of anti-CD4 therapy.     

Type D SRV-2 virus-specific CD8+ and CD4- CD8- T cells that regulate virus-induced T cell proliferation in Celebes macaques

These studies defined SRV-2 envelope peptides 96-102, 127-152, and 233-249 as T cell epitopes that induce significant T cell proliferation. Peripheral blood lymphocytes of Celebes macaques (Macaca nigra) exposed to SRV-2 and currently virus^- antibody⁺, cultured with SRV-2 virus show strongly suppressed T cell responses and have two immunoregulatory T cell populations.     

Human CD4+ T cells displaying viral epitopes elicit a functional virus-specific memory CD8+ T cell response

The Ag-specific cellular recall response to herpes virus infections is characterized by a swift recruitment of virus-specific memory T cells. Rapid activation is achieved through formation of the immunological synapse and supramolecular clustering of signal molecules at the site of contact. During the formation of the immunological synapse, epitope-loaded MHC molecules are transferred via trogocytosis from APCs to T cells, enabling the latter to function as Ag-presenting T cells (T-APCs). The contribution of viral epitope expressing T-APCs in the regulation of the herpes virus-specific CD8+ T cell memory response remains unclear. Comparison of CD4+ T-APCs with professional APCs such as Ag-presenting CD40L-activated B cells (CD40B-APCs) demonstrated reduced levels of costimulatory ligands. Despite the observed differences, CD4+ T-APCs are as potent as CD40B-APCs in stimulating herpes virus-specific CD8+ T cells resulting in a greater than 35-fold expansion of CD8+ T cells specific for dominant and subdominant viral epitopes. Virus-specific CD8+ T cells generated by CD4+ T-APCs or CD40B-APCs showed both comparable effector function such as specific lysis of targets and cytokine production and also did not differ in their phenotype after expansion. These results indicate that viral epitope presentation by Ag-specific CD4+ T cells may contribute to the rapid recruitment of virus-specific memory CD8+ T cells during a viral recall response.     

Human immunodeficiency virus-specific CD8(+) T cells in human breast milk

Breast-feeding infants of human immunodeficiency virus (HIV)-infected women ingest large amounts of HIV, but most escape infection. While the factors affecting transmission risk are poorly understood, HIV-specific cytotoxic T-lymphocyte (CTL) responses play a critical role in controlling HIV levels in blood. We therefore investigated the ability of breast milk cells (BMC) from HIV-infected women from the United States and Zambia to respond to HIV-1 peptides in a gamma interferon enzyme-linked immunospot assay. All (n = 11) HIV-infected women had responses to pools of Gag peptide (range, 105 to 1,400 spot-forming cells/million; mean = 718), 8 of 11 reacted to Pol, 7 reacted to Nef, and 2 of 5 reacted to Env. Conversely, of four HIV-negative women, none responded to any of the tested HIV peptide pools. Depletion and tetramer staining studies demonstrated that CD8(+) T cells mediated these responses, and a chromium-release assay showed that these BMC were capable of lysing target cells in an HIV-specific manner. These data demonstrate the presence of HIV-specific major histocompatibility complex class I-restricted CD8(+) CTLs in breast milk. Their presence suggests a role in limiting transmission and provides a rationale for vaccine strategies to enhance these responses.     

Suppression of virus-specific antibody production by CD8+ class I-restricted antiviral cytotoxic T cells in vivo.

The question of whether virus-induced immunosuppression includes the antibody response against the infecting virus itself was evaluated in a model situation. Transgenic mice expressing the T-cell receptor (TCR) specific for peptide 32-42 of lymphocytic choriomeningitis virus (LCMV) glycoprotein 1 presented by Db reacted with a strong transgenic cytotoxic T-lymphocyte (CTL) response starting on day 3 after infection with a high dose (10(6) PFU intravenously [i.v.]) of the WE strain of LCMV (LCMV-WE); LCMV-specific antibody production in the spleen was suppressed in these mice. Low-dose (10(2) PFU i.v.) infection resulted in an antiviral antibody response comparable to that of the transgene-negative littermates. The induction of suppression of LCMV-specific antibody responses was specifically mediated by CD8+ TCR transgenic CTLs, since the LCMV-8.7 variant virus (which is not recognized by transgenic TCR-expressing CTLs because of a point mutation) did not induce suppression. In addition, treatment with CD8 monoclonal antibody in vivo abrogated suppression. Once suppression had been established, it was found to be nonspecific. The abrogation of antibody responses depended on the relative kinetics of the antibody response involved and the kinetics of the anti-LCMV CTL response. Analysis of T- and B-cell subpopulations showed no significant changes, but immunohistochemical analysis of spleens revealed extensive destruction of follicular organization in lymphoid tissue by day 4 in transgenic mice infected with LCMV-WE but not in those infected with the CTL escape mutant LCMV-8.7. Impairment of antigen presentation rather than of T or B cells was also suggested by adoptive transfer experiments, showing that transferred infected macrophages may improve the anti-LCMV antibody response in LCMV-immunosuppressed transgenic recipients; also, T and B cells from suppressed transgenic mice did respond in irradiated and virus-infected nontransgenic mice with antibody formation to LCMV. Such virus-triggered, T-cell-mediated immunopathology causing the suppression of B cells and of protective antibody responses, including those against the infecting virus itself, may permit certain viruses to establish persistent infections.     

Regulation of cytokine production by virus-specific CD8 T cells in the lungs

Inflammation and the elimination of infected host cells during an immune response often cause local tissue injury and immunopathology, which can disrupt the normal functions of tissues such as the lung. Here, we show that both virus-induced inflammation and the host tissue environment combine to influence the capacity of virus-specific CD4 and CD8 T cells to produce cytokines in various tissues. Decreased production of cytokines, such as IFN-γ and TNF-α, by antigen-specific T cells is more pronounced in peripheral tissues, such as the lung and kidney, than in secondary lymphoid organs, such as the spleen or lymph nodes. We also demonstrate that tissues regulate cytokine production by memory T cells independently of virus infection, as memory T cells that traffic into the lungs of naïve animals exhibit a reduced ability to produce cytokines following direct ex vivo peptide stimulation. Furthermore, we show that cytokine production by antigen-specific memory CD4 and CD8 T cells isolated from the lung parenchyma can be rescued by stimulation with exogenous peptide-pulsed antigen-presenting cells. Our results suggest that the regulation of T-cell cytokine production by peripheral tissues may serve as an important mechanism to prevent immunopathology and preserve normal tissue function.     

Hidden epitopes emerge in secondary influenza virus-specific CD8+ T cell responses

Influenza A virus-specific CD8+ T cell responses in H2(b) mice are characterized by reproducible hierarchies. Compensation by the D(b)PB1-F2(62) epitope is apparent following infection with a variant H3N2 virus engineered to disrupt the prominent D(b)NP(366) and D(b)PA(224) epitopes (a double knockout or DKO). Analysis with a "triple" knockout (TKO) virus, which also compromises D(b)PB1-F2(62), did not reveal further compensation to the known residual, minor, and predicted epitopes. However, infection with this deletion mutant apparently switched protective immunity to an alternative Ab-mediated pathway. As expected, TKO virus clearance was significantly delayed in Ab-deficient MHC class II(-/-) and Ig(-/-) mice, which were much more susceptible following primary, intranasal infection with the TKO, but not DKO, virus. CD8+ T cell compensation was detected in DKO, but not TKO, infection of Ig-deficient mice, suggestive of cooperation among CD8+ T cell responses. However, after priming with a TKO H1N1 mutant, MHC II(-/-) mice survived secondary intranasal exposure to the comparable H3N2 TKO virus. Such prime/challenge experiments with the DKO and TKO viruses allowed the emergence of two previously unknown epitopes. The contrast between the absence of compensatory effect following primary exposure and the substantial clonal expansion after secondary challenge suggests that the key factor limiting the visibility of these "hidden" epitopes may be very low naive T cell precursor frequencies. Overall, these findings suggest that vaccine approaches using virus vectors to deliver an Ag may be optimized by disrupting key peptides in the normal CD8+ T cell response associated with common HLA types.     

Enhancement of suboptimal CD8 cytotoxic T cell effector function in vivo using antigen-specific CD80 defective T cells

T cell upregulation of B7 molecules CD80 and CD86 limits T cell expansion in immunodeficient hosts; however, the relative roles of CD80 separate from CD86 on CD4 versus CD8 T cells in a normal immune system are not clear. To address this question, we used the parent-into-F1 (P→F1) murine model of graft-versus-host disease and transferred optimal and suboptimal doses of CD80 and/or CD86 knockout (KO) T cells into normal F1 hosts. Enhanced elimination of host B cells by KO T cells was observed only at suboptimal donor cell doses and was greatest for CD80 KO→F1 mice. Wild-type donor cells exhibited peak CD80 upregulation at day 10; CD80 KO donor cells exhibited greater peak (day 10) donor T cell proliferation and CD8 T cell effector CTL numbers versus wild-type→F1 mice. Fas or programmed cell death-1 upregulation was normal as was homeostatic contraction of CD80 KO donor cells from days 12-14. Mixing studies demonstrated that maximal host cell elimination was seen when both CD4 and CD8 T cells were CD80 deficient. These results indicate an important role for CD80 upregulation on Ag-activated CD4 and CD8 T cells in limiting expansion of CD8 CTL effectors as part of a normal immune response. Our results support further studies of therapeutic targeting of CD80 in conditions characterized by suboptimal CD8 effector responses.     

Cutting edge: rapid in vivo CTL activity by polyoma virus-specific effector and memory CD8+ T cells

For viruses that establish persistent infection, continuous immunosurveillance by effector-competent antiviral CD8(+) T cells is likely essential for limiting viral replication. Although it is well documented that virus-specific memory CD8(+) T cells synthesize cytokines after short term in vitro stimulation, there is limited evidence that these T cells exhibit cytotoxicity, the dominant antiviral effector function. Here, we show that antiviral CD8(+) T cells in mice acutely infected by polyoma virus, a persistent mouse pathogen, specifically eliminate viral peptide-pulsed donor spleen cells within minutes after adoptive transfer and do so via a perforin-dependent mechanism. Antiviral memory CD8(+) T cells were similarly capable of rapidly mobilizing potent Ag-specific cytotoxic activity in vivo. These findings strongly support the concept that a cytotoxic effector-memory CD8(+) T cell population operates in vivo to control this persistent viral infection.     

Ex vivo expansion of human CD8+ T cells using autologous CD4+ T cell help

Background

Using in vivo mouse models, the mechanisms of CD4⁺ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4⁺ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4⁺ T cell help of CD8⁺ T cell proliferation using a novel in vitro model.

Methods/Principal Findings

We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3⁺ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4⁺ T cells was associated with significantly improved CD8⁺ T cell expansion in vitro. The CD4⁺ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4⁺ T cell help of CD8⁺ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8⁺ T cells.

Conclusions

We have developed an in vitro model that advances our understanding of the immunobiology of human CD4⁺ T cell help of CD8⁺ T cells. Our data suggests that human CD4⁺ T cell help can be leveraged to expand CD8⁺ T cells in vitro.     

Ex vivo phenotype and frequency of influenza virus-specific CD4 memory T cells

Recent advances in class II tetramer staining technology have allowed reliable direct ex vivo visualization of antigen-specific CD4 T cells. In order to define the frequency and phenotype of a prototype response to a nonpersistent pathogen, we have used such techniques to analyze influenza virus-specific memory CD4 T cells directly from blood. These responses are stably detectable ex vivo at low frequencies (range, 0.00012 to 0.0061% of CD4 T cells) and display a distinct "central memory" CD62L(+) phenotype.     

OX40 ligation of CD4+ T cells enhances virus-specific CD8+ T cell memory responses independently of IL-2 and CD4+ T regulatory cell inhibition

We have previously shown that CD4(+) T cells are required to optimally expand viral-specific memory CD8(+) CTL responses using a human dendritic cell-T cell-based coculture system. OX40 (CD134), a 50-kDa transmembrane protein of the TNFR family, is expressed primarily on activated CD4(+) T cells. In murine models, the OX40/OX40L pathway has been shown to play a critical costimulatory role in dendritic cell/T cell interactions that may be important in promoting long-lived CD4(+) T cells, which subsequently can help CD8(+) T cell responses. The current study examined whether OX40 ligation on ex vivo CD4(+) T cells can enhance their ability to "help" virus-specific CTL responses in HIV-1-infected and -uninfected individuals. OX40 ligation of CD4(+) T cells by human OX40L-IgG1 enhanced the ex vivo expansion of HIV-1-specific and EBV-specific CTL from HIV-1-infected and -uninfected individuals, respectively. The mechanism whereby OX40 ligation enhanced help of CTL was independent of the induction of cytokines such as IL-2 or any inhibitory effect on CD4(+) T regulatory cells, but was associated with a direct effect on proliferation of CD4(+) T cells. Thus, OX40 ligation on CD4(+) T cells represents a potentially novel immunotherapeutic strategy that should be investigated to treat and prevent persistent virus infections, such as HIV-1 infection.