Pichinde Virus Research Articles

Immunosuppressive arenaviral exoribonuclease

Arenaviruses cause up to 500,000 zoonotic infections per year in endemic areas of Africa and South America and can lead to severe and lethal hemorrhagic fever (HF) symptoms [1]. Currently, there is no specific antiviral drug or vaccine available for the treatment of these infections, with the exception of the Candid #1 vaccine that has successfully been used to prevent infections of Junin virus in the endemic areas of Argentina. Progression and severity of arenaviral HF disease generally correlates with viremia and host immune suppression, the molecular mechanisms of which have only recently been revealed [2]. Mounting an early and effective induction of the host innate-immune response to the infection, which is followed by robust cell-mediated responses, is thought to be essential to control the outcome of the infection. Many arenaviruses, especially those belonging to the Old World virus group, have developed different strategies to effectively suppress the host immune responses to infections. Our laboratory has been involved in characterizing the molecular mechanisms behind innate immune suppression mediated by arenaviral nucleoprotein (NP). Our high-resolution structure of the Lassa virus (LASV) NP reveals a conserved DEDDh exonuclease in its C terminal domain [3], which can degrade double-stranded RNA that would normally trigger the type I interferons (IFN) signaling pathway upon viral infections. NP has also been shown to interfere with the proper functions of several factors in the RIG-I-like receptor (RLR) pathway, namely IKKe to prevent IRF3 activation, and the Nuclear Factor Kappa B (NF-κB) pathway, and therefore suppressing the production of IFN-β and potentially other cytokines [4]. Initial studies using NPs expressed from plasmid DNAs have attributed the non-pathogenic nature of the Tacaribe virus (TCRV) to a change of amino acids around the DEDDh exonuclease site and the observed lack of IRF3 inhibition. Our efforts to resequence TCRV and to perform a comprehensive structure-function analysis of the TCRV NP have revealed discrepancies with the GenBank deposited sequence and shown that the IFN-antagonism displayed by the NP exoribonuclease (RNase) activity is a conserved mechanism among different arenaviruses, including but not necessarily limited to TCRV, LASV and Pichinde virus (PICV) [5]. Using recombinant viruses, we have recently shown the importance of the IFN-suppressive activity of NP and its role in mediating optimal viral replication in cell culture as well as in a surrogate animal model of LASV infection that is based on PICV-infected guinea pigs [5]. Specifically, we show that mutating each of the NP's catalytic DEDDh residues to alanines results in significantly reduced levels of IFN-β inhibition and virus grow potentials in immune-competent cells, and in an attenuated phenotype of the mutant viruses in infected animals. The important role of the NP RNase function in suppressing immune response in order to favor virus replication is evidenced by the appearance of wild-type virus revertants in many of the sick animals with detectable levels of viremia [5]. Our findings are supported by recent studies with recombinant LASV carrying the NP catalytic double mutations (D389T/G392A and D389A/G392A), which lose the ability to suppress the type I IFN-induction pathway in dendritic cells or macrophages, which are thought to be primary target cells for virus infection [6, 7]. The inability of the LASV NP catalytic mutants to suppress type I IFNs results in upregulated expression levels of major histocompatibility complex (MHC) class I gene pathways as well as important macrophage-derived cytokines that activate natural killer (NK) cells to participate in the direct killing of the infected cells. Thus, the proper function of the NP RNase appears to be essential in suppressing the early innate immune response, which directly correlates with the priming of cellular immunity and therefore impacts virus clearance in vivo. Taken together, studies conducted in our laboratory as well as by other laboratories have provided strong evidence to support the role of the arenaviral NP exoribonuclease function in mediating immune suppression upon viral infections. It is important to highlight the fact that this is the first time that a viral exoribonuclease function has been attributed to mediating host immune evasion. Because there is currently no specific antiviral therapy available against arenaviruses, a better level of understanding of how arenaviruses are able to evade host immunity is warranted as it may offer new ways to evaluate future treatment options and to design and test vaccine candidates against these deadly human viral pathogens.

Differential Inhibition of Macrophage Activation by Lymphocytic Choriomeningitis Virus and Pichinde Virus Is Mediated by the Z Protein N-Terminal Domain.

Several arenavirus pathogens, such as Lassa and Junin viruses, inhibit macrophage activation, the molecular mechanism of which is unclear. We show that lymphocytic choriomeningitis virus (LCMV) can also inhibit macrophage activation, in contrast to Pichinde and Tacaribe viruses, which are not known to naturally cause human diseases. Using a recombinant Pichinde virus system, we show that the LCMV Z N-terminal domain (NTD) mediates the inhibition of macrophage activation and immune functions.

Increased Immune Response Variability during Simultaneous Viral Coinfection Leads to Unpredictability in CD8 T Cell Immunity and Pathogenesis.

T cell memory is usually studied in the context of infection with a single pathogen in naive mice, but how memory develops during a coinfection with two pathogens, as frequently occurs in nature or after vaccination, is far less studied. Here, we questioned how the competition between immune responses to two viruses in the same naive host would influence the development of CD8 T cell memory and subsequent disease outcome upon challenge. Using two different models of coinfection, including the well-studied lymphocytic choriomeningitis (LCMV) and Pichinde (PICV) viruses, several differences were observed within the CD8 T cell responses to either virus. Compared to single-virus infection, coinfection resulted in substantial variation among mice in the size of epitope-specific T cell responses to each virus. Some mice had an overall reduced number of virus-specific cells to either one of the viruses, and other mice developed an immunodominant response to a normally subdominant, cross-reactive epitope (nucleoprotein residues 205 to 212, or NP205). These changes led to decreased protective immunity and enhanced pathology in some mice upon challenge with either of the original coinfecting viruses. In mice with PICV-dominant responses, during a high-dose challenge with LCMV clone 13, increased immunopathology was associated with a reduced number of LCMV-specific effector memory CD8 T cells. In mice with dominant cross-reactive memory responses, during challenge with PICV increased immunopathology was directly associated with these cross-reactive NP205-specific CD8 memory cells. In conclusion, the inherent competition between two simultaneous immune responses results in significant alterations in T cell immunity and subsequent disease outcome upon reexposure. Combination vaccines and simultaneous administration of vaccines are necessary to accommodate required immunizations and maintain vaccination rates. Antibody responses generally correlate with protection and vaccine efficacy. However, live attenuated vaccines also induce strong CD8 T cell responses, and the impact of these cells on subsequent immunity, whether beneficial or detrimental, has seldom been studied, in part due to the lack of known T cell epitopes to vaccine viruses. We questioned if the inherent increased competition and stochasticity between two immune responses during a simultaneous coinfection would significantly alter CD8 T cell memory in a mouse model where CD8 T cell epitopes are clearly defined. We show that some of the coinfected mice have sufficiently altered memory T cell responses that they have decreased protection and enhanced immunopathology when reexposed to one of the two viruses. These data suggest that a better understanding of human T cell responses to vaccines is needed to optimize immunization strategies.

Heterologous protective immunity can lead to impaired activation, memory differentiation and inflation of new naive responses (VIR2P.1170)

Abstract Activation of crossreactive memory CD8 T cells leads to heterologous protective immunity. Here we question whether early activation of crossreactive memory CD8 T cells alters the activation of new naïve CD8 responses to the second virus. Pichinde virus (PICV) and lymphocytic choriomeningitis virus (LCMV) contain a crossreactive usually subdominant epitope, NP205, which differs in 6 of 8 amino acids. During PICV infection of LCMV-immune mice the NP205 memory response become immunodominant and mediates protective immunity. PICV is cleared early and does not persist. The usually dominant non-crossreactive new naïve PICV NP38 CD8 T cell response appears to be subdominant day 7 post infection. However, surprisingly over the next 100 days post infection the NP38 response continues to gradually expand and then stabilizes. These NP38-specific CD8 T cells appear to arrest in an intermediate effector T cell phenotype (KLRG1low, CD62Llow IL-7Rhi), with altered functionality (decreased TNFα production). They appear to have altered expression of apoptotic genes on gene array. This NP38 inflationary response did not occur when mice were immunized with an LCMV mutant that did not present the crossreactive NP205 epitope. Our data shows that the strong crossreactive response kills DC’s expressing NP205 early (day 2-3) in PICV infection. This may result in an early termination of antigen-presentation leading to inadequate activation of the naïve NP38 response and inflation.

In Vitro and In Vivo Characterizations of Pichinde Viral Nucleoprotein Exoribonuclease Functions

Arenaviruses cause severe hemorrhagic fever diseases in humans, and there are limited preventative and therapeutic measures against these diseases. Previous structural and functional analyses of arenavirus nucleoproteins (NPs) revealed a conserved DEDDH exoribonuclease (RNase) domain that is important for type I interferon (IFN) suppression, but the biological roles of the NP RNase in viral replication and host immune suppression have not been well characterized. Infection of guinea pigs with Pichinde virus (PICV), a prototype arenavirus, can serve as a surrogate small animal model for arenavirus hemorrhagic fevers. In this report, we show that mutation of each of the five RNase catalytic residues of PICV NP diminishes the IFN suppression activity and slightly reduces the viral RNA replication activity. Recombinant PICVs with RNase catalytic mutations can induce high levels of IFNs and barely grow in IFN-competent A549 cells, in sharp contrast to the wild-type (WT) virus, while in IFN-deficient Vero cells, both WT and mutant viruses can replicate at relatively high levels. Upon infection of guinea pigs, the RNase mutant viruses stimulate strong IFN responses, fail to replicate productively, and can become WT revertants. Serial passages of the RNase mutants in vitro can also generate WT revertants. Thus, the NP RNase function is essential for the innate immune suppression that allows the establishment of a productive early viral infection, and it may be partly involved in the process of viral RNA replication. Arenaviruses, such as Lassa, Lujo, and Machupo viruses, can cause severe and deadly hemorrhagic fever diseases in humans, and there are limited preventative and treatment options against these diseases. Development of broad-spectrum antiviral drugs depends on a better mechanistic understanding of the conserved arenavirus proteins in viral infection. The nucleoprotein (NPs) of all arenaviruses carry a unique exoribonuclease (RNase) domain that has been shown to be critical for the suppression of type I interferons. However, the functional roles of the NP RNase in arenavirus replication and host immune suppression have not been characterized systematically. Using a prototype arenavirus, Pichinde virus (PICV), we characterized the viral growth and innate immune suppression of recombinant RNase-defective mutants in both cell culture and guinea pig models. Our study suggests that the NP RNase plays an essential role in the suppression of host innate immunity, and possibly in viral RNA replication, and that it can serve as a novel target for developing antiviral drugs against arenavirus pathogens.

Characterization of the Host Response to Pichinde Virus Infection in the Syrian Golden Hamster by Species-Specific Kinome Analysis

The Syrian golden hamster has been increasingly used to study viral hemorrhagic fever (VHF) pathogenesis and countermeasure efficacy. As VHFs are a global health concern, well-characterized animal models are essential for both the development of therapeutics and vaccines as well as for increasing our understanding of the molecular events that underlie viral pathogenesis. However, the paucity of reagents or platforms that are available for studying hamsters at a molecular level limits the ability to extract biological information from this important animal model. As such, there is a need to develop platforms/technologies for characterizing host responses of hamsters at a molecular level. To this end, we developed hamster-specific kinome peptide arrays to characterize the molecular host response of the Syrian golden hamster. After validating the functionality of the arrays using immune agonists of defined signaling mechanisms (lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α), we characterized the host response in a hamster model of VHF based on Pichinde virus (PICV(1)) infection by performing temporal kinome analysis of lung tissue. Our analysis revealed key roles for vascular endothelial growth factor (VEGF), interleukin (IL) responses, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, and Toll-like receptor (TLR) signaling in the response to PICV infection. These findings were validated through phosphorylation-specific Western blot analysis. Overall, we have demonstrated that hamster-specific kinome arrays are a robust tool for characterizing the species-specific molecular host response in a VHF model. Further, our results provide key insights into the hamster host response to PICV infection and will inform future studies with high-consequence VHF pathogens.

The Z proteins of pathogenic but not nonpathogenic arenaviruses inhibit RIG-I-like receptor-dependent interferon production.

Arenavirus pathogens cause a wide spectrum of diseases in humans ranging from central nervous system disease to lethal hemorrhagic fevers with few treatment options. The reason why some arenaviruses can cause severe human diseases while others cannot is unknown. We find that the Z proteins of all known pathogenic arenaviruses, lymphocytic choriomeningitis virus (LCMV) and Lassa, Junin, Machupo, Sabia, Guanarito, Chapare, Dandenong, and Lujo viruses, can inhibit retinoic acid-inducible gene 1 (RIG-i) and Melanoma Differentiation-Associated protein 5 (MDA5), in sharp contrast to those of 14 other nonpathogenic arenaviruses. Inhibition of the RIG-i-like receptors (RLRs) by pathogenic Z proteins is mediated by the protein-protein interactions of Z and RLRs, which lead to the disruption of the interactions between RLRs and mitochondrial antiviral signaling (MAVS). The Z-RLR interactive interfaces are located within the N-terminal domain (NTD) of the Z protein and the N-terminal CARD domains of RLRs. Swapping of the LCMV Z NTD into the nonpathogenic Pichinde virus (PICV) genome does not affect virus growth in Vero cells but significantly inhibits the type I interferon (IFN) responses and increases viral replication in human primary macrophages. In summary, our results show for the first time an innate immune-system-suppressive mechanism shared by the diverse pathogenic arenaviruses and thus shed important light on the pathogenic mechanism of human arenavirus pathogens. We show that all known human-pathogenic arenaviruses share an innate immune suppression mechanism that is based on viral Z protein-mediated RLR inhibition. Our report offers important insights into the potential mechanism of arenavirus pathogenesis, provides a convenient way to evaluate the pathogenic potential of known and/or emerging arenaviruses, and reveals a novel target for the development of broad-spectrum therapies to treat this group of diverse pathogens. More broadly, our report provides a better understanding of the mechanisms of viral immune suppression and host-pathogen interactions.

MHC basis of T cell-dependent heterologous immunity to arenaviruses

Having a history of infection with one pathogen may sometimes provide a level of T cell-dependent protective heterologous immunity to another pathogen. This immunity was initially thought due to cross-reactive T cell epitopes, but recent work has suggested that such protective immunity can be initiated nonspecifically by the action of cytokines on memory T cells. We retested this concept using two small and well-defined arenaviruses, lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PV), and found that heterologous immunity in these systems was indeed linked to T cell epitopes and the major histocompatibility complex.

Inhibition of arenavirus infection by a glycoprotein-derived peptide with a novel mechanism.

The family Arenaviridae includes a number of viruses of public health importance, such as the category A hemorrhagic fever viruses Lassa virus, Junin virus, Machupo virus, Guanarito virus, and Sabia virus. Current chemotherapy for arenavirus infection is limited to the nucleoside analogue ribavirin, which is characterized by considerable toxicity and treatment failure. Using Pichinde virus as a model arenavirus, we attempted to design glycoprotein-derived fusion inhibitors similar to the FDA-approved anti-HIV peptide enfuvirtide. We have identified a GP2-derived peptide, AVP-p, with antiviral activity and no acute cytotoxicity. The 50% inhibitory dose (IC50) for the peptide is 7 μM, with complete inhibition of viral plaque formation at approximately 20 μM, and its antiviral activity is largely sequence dependent. AVP-p demonstrates activity against viruses with the Old and New World arenavirus viral glycoprotein complex but not against enveloped viruses of other families. Unexpectedly, fusion assays reveal that the peptide induces virus-liposome fusion at neutral pH and that the process is strictly glycoprotein mediated. As observed in cryo-electron micrographs, AVP-p treatment causes morphological changes consistent with fusion protein activation in virions, including the disappearance of prefusion glycoprotein spikes and increased particle diameters, and fluorescence microscopy shows reduced binding by peptide-treated virus. Steady-state fluorescence anisotropy measurements suggest that glycoproteins are destabilized by peptide-induced alterations in viral membrane order. We conclude that untimely deployment of fusion machinery by the peptide could render virions less able to engage in on-pathway receptor binding or endosomal fusion. AVP-p may represent a potent, highly specific, novel therapeutic strategy for arenavirus infection. Because the only drug available to combat infection by Lassa virus, a highly pathogenic arenavirus, is toxic and prone to treatment failure, we identified a peptide, AVP-p, derived from the fusion glycoprotein of a nonpathogenic model arenavirus, which demonstrates antiviral activity and no acute cytotoxicity. AVP-p is unique among self-derived inhibitory peptides in that it shows broad, specific activity against pseudoviruses bearing Old and New World arenavirus glycoproteins but not against viruses from other families. Further, the peptide's mechanism of action is highly novel. Biochemical assays and cryo-electron microscopy indicate that AVP-p induces premature activation of viral fusion proteins through membrane perturbance. Peptide treatment, however, does not increase the infectivity of cell-bound virus. We hypothesize that prematurely activated virions are less fit for receptor binding and membrane fusion and that AVP-p may represent a viable therapeutic strategy for arenavirus infection.

Properties of virus-induced NK cells correlating with their ability to kill activated CD4 T cells (IRM7P.489)

Abstract Activated natural killer (NK) cells induced during viral infections of mice can regulate adaptive immunity by lysing activated CD4 T cells. This seems to be a general property of interferon-inducing viruses, but the efficiency of this process is in part dependent on the nature of the instigating virus. Here we examined the differential expression of activating and inhibitory receptors on NK cells from lymphocytic choriomeningitis virus (LCMV) and vaccinia virus (VACV)-infected mice, because NK cells from VACV-infected mice were far less efficient at killing activated CD4 T cells than those from LCMV-infected mice. Relative to expression levels on LCMV-activated NK cells, NKG2A expression was higher in VACV-induced NK cells while NKG2D was lower. NK cells activated by infections with LCMV, Pichinde virus (PV), mouse hepatitis virus (MHV), and murine cytomegalovirus (MCMV) displayed similar expression patterns for all of the examined receptors, whereas VACV-induced cells had a distinct expression pattern. Another major difference between VACV-induced NK cells and NK cells activated by the other viruses was in the expression of granzyme B, an essential component of NK cell-mediated cytolysis. VACV-induced NK cells expressed granzyme B at lower levels than NK cells from mice infected with LCMV, PV, MHV A59, or MCMV. These results indicate that the NK cell receptor expression and the cytolytic activity of NK cells can vary with the infecting virus.

Mechanisms underlying specificity of natural killer cell regulation of adaptive immunity (IRC8P.485)

Abstract Viral persistence is determined in part by the regulatory activities of murine natural killer (NK) cells, including the lysis of activated CD4 T cells. We now find that NK cells also impair virus-specific memory T cell responses [and repress humoral immunity (separate abstract)] after acute Pichinde or lymphocytic choriomeningitis virus infections. Hence, limiting NK cell inhibition of adaptive immunity during vaccination could enhance long-lived protective immunity. To enable selective targeting of this NK cell activity in a vaccine, the molecular mechanisms of such suppression must be ascertained. Thus, we have explored changes in the expression of NK cell receptors (NKR) and their ligands (NKRL) on both NK cells and target T cells after infection. This analysis revealed that: 1) suppression of adaptive immunity corresponded with reduced inhibitory NKR expression on NK cells; 2) archetypical activating NKR such as NKG2D were dispensable in our system; and 3) susceptibility of activated CD4 T cells to NK cell lysis correlated with reduced expression of inhibitory NKRL (e.g. CD48) and corresponding higher levels of activating NKRL (e.g. Ly108), than were present on NK cell-resistant CD8 T cells. Further, the selectivity of NK cell killing of different T cells could be deregulated by altering NKR/NKRL expression. Our findings may facilitate development of innovative strategies to harness or inhibit NK cell lysis of T cells in the context of vaccination or immune therapy.

Participation of eIF4F complex in Junin virus infection: blockage of eIF4E does not impair virus replication

Translation efficiency of viral mRNAs is a key factor defining both cytopathogenicity and virulence of viruses, which are entirely dependent on the cellular translation machinery to synthesize their proteins. This dependence has led them to develop different translational reprogramming strategies to ensure viral mRNAs can effectively compete with cellular mRNAs. Junin virus (JUNV) is a member of the family Arenaviridae, whose mRNAs are capped but not polyadenylated. In this work we evaluated the relevance to JUNV replication of the main components of the eIF4F complex: eIF4A, eIF4GI and eIF4E. We found the viral nucleoprotein (N) of JUNV colocalized with eIF4A and eIF4GI but not with eIF4E. Moreover, N could be immunoprecipitated in association with eIF4A and eIF4GI but not with eIF4E. Accordingly, functional impairment of eIF4A as well as eIF4GI reduced JUNV multiplication. By contrast, inhibition of eIF4E did not show a significant effect on JUNV protein synthesis. A similar situation was observed for another two members of arenaviruses: Tacaribe (TCRV) and Pichinde (PICV) viruses. Finally, the nucleoproteins of JUNV, TCRV and PICV were able to interact with 7 methyl-guanosine (cap), suggesting that the independence of JUNV multiplication on eIF4E, the cap-binding protein, may be due to the replacement of this factor by N protein.

Identification of Critical Amino Acids within the Nucleoprotein of Tacaribe Virus Important for Anti-interferon Activity

The arenavirus nucleoprotein (NP) can suppress induction of type I interferon (IFN). This anti-IFN activity is thought to be shared by all arenaviruses with the exception of Tacaribe virus (TCRV). To identify the TCRV NP amino acid residues that prevent its IFN-countering ability, we created a series of NP chimeras between residues of TCRV NP and Pichinde virus (PICV) NP, an arenavirus NP with potent anti-IFN function. Chimera NP analysis revealed that a minimal four amino acid stretch derived from PICV NP could impart efficient anti-IFN activity to TCRV NP. Strikingly, the TCRV NP gene cloned and sequenced from viral stocks obtained through National Institutes of Health Biodefense and Emerging Infections (BEI) resources deviated from the reference sequence at this particular four-amino acid region, GPPT (GenBank KC329849) versus DLQL (GenBank NC004293), respectively at residues 389-392. When efficiently expressed in cells through codon-optimization, TCRV NP containing the GPPT residues rescued the antagonistic IFN function. Consistent with cell expression results, TCRV infection did not stimulate an IFNβ response early in infection in multiple cells types (e.g. A549, P388D1), and IRF-3 was not translocated to the nucleus in TCRV-infected A549 cells. Collectively, these data suggest that certain TCRV strain variants contain the important NP amino acids necessary for anti-IFN activity.

Characterization of virulence-associated determinants in the envelope glycoprotein of Pichinde virus

We use a small animal model, based on guinea pigs infected with a non-pathogenic Pichinde virus (PICV), to understand the virulence mechanisms of arenavirus infections in the hosts. PICV P2 strain causes a mild febrile reaction in guinea pigs, while P18 causes severe disease with clinical and pathological features reminiscent of Lassa hemorrhagic fever in humans. The envelope glycoproteins (GPC) of P2 and P18 viruses differ at positions 119, 140, and 164, all localized to the receptor-binding G1 subunit. We found that lentiviral pseudotyped virions (VLPs) bearing P18 GPC show more efficient cell entry than those with P2 GPC, and that the E140 residue plays a critical role in this process. Infection of guinea pigs with the recombinant viruses containing the E140K change demonstrated that E140 of GPC is a necessary virulence determinant of P18 infections, possibly by enhancing the ability of virus to enter target cells.

Biological Roles and Functional Mechanisms of Arenavirus Z Protein in Viral Replication

Arenaviruses can cause severe hemorrhagic fever diseases in humans, with limited prophylactic or therapeutic measures. A small RING-domain viral protein Z has been shown to mediate the formation of virus-like particles and to inhibit viral RNA synthesis, although its biological roles in an infectious viral life cycle have not been directly addressed. By taking advantage of the available reverse genetics system for a model arenavirus, Pichinde virus (PICV), we provide the direct evidence for the essential biological roles of the Z protein's conserved residues, including the G2 myristylation site, the conserved C and H residues of RING domain, and the poorly characterized C-terminal L79 and P80 residues. Dicodon substitutions within the late (L) domain (PSAPPYEP) of the PICV Z protein, although producing viable mutant viruses, have significantly reduced virus growth, a finding suggestive of an important role for the intact L domain in viral replication. Further structure-function analyses of both PICV and Lassa fever virus Z proteins suggest that arenavirus Z proteins have similar molecular mechanisms in mediating their multiple functions, with some interesting variations, such as the role of the G2 residue in blocking viral RNA synthesis. In summary, our studies have characterized the biological roles of the Z protein in an infectious arenavirus system and have shed important light on the distinct functions of its domains in virus budding and viral RNA regulation, the knowledge of which may lead to the development of novel antiviral drugs.

Co-infection alters CD8 T cell immunity and immunoprotection (105.27)

Abstract Co-infection with multiple pathogens can result in increased pathology and difficulties with diagnosis and treatment. Moreover, interference between co-administered vaccines has resulted in lower antibody production. However, the effect of co-infections on T cell responses remains poorly understood. We have shown that sequential infection with unrelated viruses can alter T cell responses and disease outcome. This is defined as heterologous immunity, which is mediated by crossreactive T cells. We questioned if co-infection with two viruses containing a crossreactive epitope would alter the immune response and disease outcome upon rechallenge. Compared to mice infected with a single virus, rechallenged of mice co-infected with lymphocytic choriomenigitis (LCMV) and Pichinde viruses (PV) had a loss of immunoprotection and enhanced immunopathology. Each viral rechallenge identified a different potential mechanism to explain these changes. First, co-infection resulted in fewer LCMV-specific effector-like memory CD8 T cells leading to decreased protection and enhanced liver pathology. Secondly, co-infection caused some mice to have inappropriately enhanced crossreactive responses, which contributed to increased immunopathology. As combined vaccines or simultaneous immunizations are also co-infections, our data would suggest that T cell responses should be examined in the context of vaccine development.

Immune regulation by natural killer cells through lysis of activated T cells (168.4)

Abstract We have previously shown that natural killer (NK) cells can influence viral pathogenesis either by directly lysing virus-infected cells or through lysis of activated CD4 T cells. The latter phenomenon contributed to an inhibition of anti-viral T cells that favored viral persistence and host survival rather than lethal pathology during lymphocytic choriomeningitis virus (LCMV) infection. Infections with unrelated viruses, including Pichinde virus (PV), or inoculation with the type I IFN inducer, polyI:C, also triggered NK cell lysis of activated CD4 T cells. This lysis was coincident with poor control of PV infection and diminished PV-specific memory T cell responses, suggesting that such NK cell immunoregulation is a general principle of virus infection. LCMV infection was unable to induce NK cell lysis of activated T cells in IFN receptor (IFNAR)-deficient mice, implicating type I IFN in stimulation of this NK cell activity. Expression of the CD48, the ligand for the inhibitory NK cell receptor 2B4/CD244, was greater on NK cell-resistant CD8 T cells than their CD4 counterparts. However, genetic deletion of 2B4 facilitated lysis of activated CD8 T cells, and was associated with impaired clearance of both acute and chronic virus infections. Thus, type I IFN expression during virus infection or vaccination stimulates a 2B4/CD48-regulated, NK cell-mediated lysis of anti-viral T cells which impairs viral control and immune memory.

Loss of Anti-Viral Immunity by Infection with a Virus Encoding a Cross-Reactive Pathogenic Epitope

T cell cross-reactivity between different strains of the same virus, between different members of the same virus group, and even between unrelated viruses is a common occurrence. We questioned here how an intervening infection with a virus containing a sub-dominant cross-reactive T cell epitope would affect protective immunity to a previously encountered virus. Pichinde virus (PV) and lymphocytic choriomeningitis virus (LCMV) encode subdominant cross-reactive NP205–212 CD8 T cell epitopes sharing 6 of 8 amino acids, differing only in the MHC anchoring regions. These pMHC epitopes induce cross-reactive but non-identical T cell receptor (TCR) repertoires, and structural studies showed that the differing anchoring amino acids altered the conformation of the MHC landscape presented to the TCR. PV-immune mice receiving an intervening infection with wild type but not NP205-mutant LCMV developed severe immunopathology in the form of acute fatty necrosis on re-challenge with PV, and this pathology could be predicted by the ratio of NP205-specific to the normally immunodominant PV NP38–45 -specific T cells. Thus, cross-reactive epitopes can exert pathogenic properties that compromise protective immunity by impairing more protective T cell responses.

Receptor Tyrosine Kinase Inhibitors That Block Replication of Influenza A and Other Viruses

We have previously reported that two receptor tyrosine kinase inhibitors (RTKIs), called AG879 and tyrphostin A9 (A9), can each block the replication of influenza A virus in cultured cells. In this study, we further characterized the in vitro antiviral efficacies and specificities of these agents. The 50% effective concentration (EC(50)) of each against influenza A was found to be in the high nanomolar range, and the selectivity index (SI = 50% cytotoxic concentration [CC(50)]/EC(50)) was determined to be >324 for AG879 and 50 for A9, indicating that therapeutically useful concentrations of each drug produce only low levels of cytotoxicity. Each compound showed efficacy against representative laboratory strains of both human influenza A (H1N1 or H3N2) and influenza B viruses. Importantly, no drug-resistant influenza virus strains emerged even after 25 viral passages in the presence of AG879, whereas viruses resistant to amantadine appeared after only 3 passages. AG879 and A9 each also exhibited potent inhibitory activity against a variety of other RNA and DNA viruses, including Sendai virus (Paramyxoviridae), herpes simplex virus (Herpesviridae), mouse hepatitis virus (Coronaviridae), and rhesus rotavirus (Reoviridae), but not against Pichinde virus (Arenaviridae). These results together suggest that RTKIs may be useful as therapeutics against viral pathogens, including but not limited to influenza, due to their high selectivity indices, low frequency of drug resistance, and broad-spectrum antiviral activities.

Development of Peptide-Conjugated Morpholino Oligomers as Pan-Arenavirus Inhibitors

Members of the Arenaviridae family are a threat to public health and can cause meningitis and hemorrhagic fever, and yet treatment options remain limited by a lack of effective antivirals. In this study, we found that peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) complementary to viral genomic RNA were effective in reducing arenavirus replication in cell cultures and in vivo. PPMO complementary to the Junín virus genome were designed to interfere with viral RNA synthesis or translation or both. However, only PPMO designed to potentially interfere with translation were effective in reducing virus replication. PPMO complementary to sequences that are highly conserved across the arenaviruses and located at the 5' termini of both genomic segments were effective against Junín virus, Tacaribe virus, Pichinde virus, and lymphocytic choriomeningitis virus (LCMV)-infected cell cultures and suppressed viral titers in the livers of LCMV-infected mice. These results suggest that arenavirus 5' genomic termini represent promising targets for pan-arenavirus antiviral therapeutic development.