Lymphocytic Choriomeningitis Virus Lineage V in Wood Mice, Germany.

We identified a novel lineage of lymphocytic choriomeningitis virus, tentatively named lineage V, in wood mice (Apodemus sylvaticus) from Germany. Wood mouse-derived lymphocytic choriomeningitis virus can be found across a substantially greater range than previously thought. Increased surveillance is needed to determine its geographic range and zoonotic potential.

A replicating LCMV-based vaccine for the treatment of solid tumors.

Harnessing the immune system to eradicate tumors requires identification and targeting of tumor antigens, including tumor-specific neoantigens and tumor-associated self-antigens. Tumor-associated antigens are subject to existing immune tolerance, which must be overcome by immunotherapies. Despite many novel immunotherapies reaching clinical trials, inducing self-antigen-specific immune responses remains challenging. Here, we systematically investigate viral-vector-based cancer vaccines encoding a tumor-associated self-antigen (TRP2) for the treatment of established melanomas in preclinical mouse models, alone or in combination with adoptive T cell therapy. We reveal that, unlike foreign antigens, tumor-associated antigens require replication of lymphocytic choriomeningitis virus (LCMV)-based vectors to break tolerance and induce effective antigen-specific CD8+ T cell responses. Immunization with a replicating LCMV vector leads to complete tumor rejection when combined with adoptive TRP2-specific T cell transfer. Importantly, immunization with replicating vectors leads to extended antigen persistence in secondary lymphoid organs, resulting in efficient T cell priming, which renders previously "cold" tumors open to immune infiltration and reprograms the tumor microenvironment to "hot." Our findings have important implications for the design of next-generation immunotherapies targeting solid cancers utilizing viral vectors and adoptive cell transfer.

Human cases of lymphocytic choriomeningitis virus (LCMV) infections in Hungary.

Lymphocytic choriomeningitis (LCM) is a "neglected" rodent-borne viral zoonotic disease caused by lymphocytic choriomeningitis virus (LCMV) (family Arenaviridae). The aim of this retrospective clinical and laboratory study was to detect LCMV RNA, using RT-PCR, in cerebrospinal fluid samples collected from patients with central nervous system (CNS) infections of unknown aetiology from over a 12-year period in Hungary. Between 2009 and 2020, a total of 74 cerebrospinal fluid samples were tested using an in-house LCMV-specific RT-PCR-based method at the Department of Medical Microbiology and Immunology, University of Pécs. The mean age of the 74 patients included in our study was 24 years (min. 5, max. 74), with a predominance of men (44 [59.5%]; women, 30 [40.5%]). Two (2.7%) cerebrospinal fluid samples were found to be positive for LCMV RNA by RT-PCR and sequencing. The first LCMV case was a 5-year-old preschool boy who had a hamster bite on his left-hand finger, and the second LCMV case was a 74-year-old man who was living in a village and had incipient dementia and a previous permanent functional CNS impairment. The two detected LCMV strains (MW558451 and OM648933) from the year 2020 belonged to two different genetic lineages (I and II). These two cases of CNS inflammation of unknown origin represent the first published human LCMV infections confirmed by molecular methods in Hungary.

External quality assessment of orthohantavirus and lymphocytic choriomeningitis virus molecular detection and serology in Europe, 2021.

BackgroundRodent-borne viruses such as orthohantaviruses and arenaviruses cause considerable disease burden with regional and temporal differences in incidence and clinical awareness. Therefore, it is important to regularly evaluate laboratory diagnostic capabilities, e.g. by external quality assessments (EQA).AimWe wished to evaluate the performance and diagnostic capability of European expert laboratories to detect orthohantaviruses and lymphocytic choriomeningitis virus (LCMV) and human antibody response towards orthohantaviruses.MethodsWe conducted an EQA in 2021; molecular panels consisted of 12 samples, including different orthohantaviruses (Seoul, Dobrava-Belgrade (DOBV), Puumala (PUUV) and Hantaan orthohantavirus), LCMV and negative controls. Serological panels consisted of six human serum samples reactive to PUUV, DOBV or negative to orthohantaviruses. The EQA was sent to 25 laboratories in 20 countries.ResultsThe accuracy of molecular detection of orthohantaviruses varied (50‒67%, average 62%) among 16 participating laboratories, while LCMV samples were successfully detected in all 11 participating laboratories (91-100%, average 96%). The accuracy of serological diagnosis of acute and past orthohantavirus infections was on average 95% among 20 participating laboratories and 82% in 19 laboratories, respectively. A variety of methods was used, with predominance of in-house assays for molecular tests, and commercial assays for serological ones.ConclusionSerology, the most common tool to diagnose acute orthohantavirus infections, had a high accuracy in this EQA. The molecular detection of orthohantaviruses needs improvement while LCMV detection (performed in fewer laboratories) had 95% accuracy. Further EQAs are recommended to be performed periodically to monitor improvements and challenges in the diagnostics of rodent-borne diseases.

Unleashing T cell anti-tumor immunity: new potential for 5-Nonloxytryptamine as an agent mediating MHC-I upregulation in tumors.

BACKGROUND: New therapies are urgently needed in melanoma, particularly in late-stage patients not responsive to immunotherapies and kinase inhibitors. To uncover novel potentiators of T cell anti-tumor immunity, we carried out an ex vivo pharmacological screen and identified 5-Nonyloxytryptamine (5-NL), a serotonin agonist, as increasing the ability of T cells to target tumor cells. METHODS: The pharmacological screen utilized lymphocytic choriomeningitis virus (LCMV)-primed splenic T cells and melanoma B16.F10 cells expressing the LCMV gp33 CTL epitope. In vivo tumor growth in C57BL/6 J and NSG mice, in vivo antibody depletion, flow cytometry, immunoblot, CRISPR/Cas9 knockout, histological and RNA-Seq analyses were used to decipher 5-NL's immunomodulatory effects in vitro and in vivo. RESULTS: 5-NL delayed tumor growth in vivo and the phenotype was dependent on the hosts' immune system, specifically CD8+ T cells. 5-NL's pro-immune effects were not directly consequential to T cells. Rather, 5-NL upregulated antigen presenting machinery in melanoma and other tumor cells in vitro and in vivo without increasing PD-L1 expression. Mechanistic studies indicated that 5-NL's induced MHC-I expression was inhibited by pharmacologically preventing cAMP Response Element-Binding Protein (CREB) phosphorylation. Importantly, 5-NL combined with anti-PD1 therapy showed significant improvement when compared to single anti-PD-1 treatment. CONCLUSIONS: This study demonstrates novel therapeutic opportunities for augmenting immune responses in poorly immunogenic tumors.

Cell-Intrinsic CD38 Expression Sustains Exhausted CD8+ T Cells by Regulating Their Survival and Metabolism during Chronic Viral Infection.

Acute and chronic viral infections result in the differentiation of effector and exhausted T cells with functional and phenotypic differences that dictate whether the infection is cleared or progresses to chronicity. High CD38 expression has been observed on CD8+ T cells across various viral infections and tumors in patients, suggesting an important regulatory function for CD38 on responding T cells. Here, we show that CD38 expression was increased and sustained on exhausted CD8+ T cells following chronic lymphocytic choriomeningitis virus (LCMV) infection, with lower levels observed on T cells from acute LCMV infection. We uncovered a cell-intrinsic role for CD38 expression in regulating the survival of effector and exhausted CD8+ T cells. We observed increased proliferation and function of Cd38-/- CD8+ progenitor exhausted T cells compared to those of wild-type (WT) cells. Furthermore, decreased oxidative phosphorylation and glycolytic potential were observed in Cd38-/- CD8+ T cells during chronic but not acute LCMV infection. Our studies reveal that CD38 has a dual cell-intrinsic function in CD8+ T cells, where it decreases proliferation and function yet supports their survival and metabolism. These findings show that CD38 is not only a marker of T cell activation but also has regulatory functions on effector and exhausted CD8+ T cells. IMPORTANCE Our study shows how CD38 expression is regulated on CD8+ T cells responding during acute and chronic viral infection. We observed higher CD38 levels on CD8+ T cells during chronic viral infection compared to levels during acute viral infection. Deleting CD38 had an important cell-intrinsic function in ensuring the survival of virus-specific CD8+ T cells throughout the course of viral infection. We found defective metabolism in Cd38-/- CD8+ T cells arising during chronic infection and changes in their progenitor T cell phenotype. Our studies revealed a dual cell-intrinsic role for CD38 in limiting proliferation and granzyme B production in virus-specific exhausted T cells while also promoting their survival. These data highlight new avenues for research into the mechanisms through which CD38 regulates the survival and metabolism of CD8+ T cell responses to viral infections.

Chronic LCMV infection regulates the effector T cell response by inducing the generation of less immunogenic dendritic cells.

Chronic viral infection impairs systemic immunity in the host; however, the mechanism underlying the dysfunction of immune cells in chronic viral infection is incompletely understood. In this study, we studied the lineage differentiation of hematopoietic stem cells (HSCs) during chronic viral infection to elucidate the changes in dendritic cell (DC) differentiation and subsequent impact on T cell functionality using a chronic lymphocytic choriomeningitis virus (LCMV) infection model. We first investigated the lineage differentiation of HSCs in the bone marrow (BM) to elucidate the modulation of immune cell differentiation and found that the populations highly restrained in their differentiation were common myeloid progenitors (CMPs) and common dendritic cell progenitors (CDPs). Of interest, the main immune cells infected with LCMV Clone 13 (CL13) in the BM were CD11b/c+ myeloid DCs. We next characterized CD11b+ DCs that differentiated during chronic LCMV infection. These DCs displayed a less immunogenic phenotype than DCs in naive or acutely infected mice, showing low expression of CD80 but high expression of PD-L1, B7-H4, IDO, TGF-ß, and IL-10. Consequently, these CD11b+ DCs induced less effective CD8+ T cells and more Foxp3+ regulatory T (Treg) cells. Furthermore, CD11b+ DCs generated during CL13 infection could not induce effective CD8+ T cells specific to the antigens of newly invading pathogens. Our findings demonstrate that DCs generated from the BM during chronic viral infection cannot activate fully functional effector CD8+ T cells specific to newly incoming antigens as well as persistent antigens themselves, suggesting a potential cause of the functional alterations in the T cell immune response during chronic viral infection.

Pseudotyped Viruses for Mammarenavirus.

Mammarenaviruses are classified into New World arenaviruses (NW) and Old World arenaviruses (OW). The OW arenaviruses include the first discovered mammarenavirus-lymphocytic choriomeningitis virus (LCMV) and the highly lethal Lassa virus (LASV). Mammarenaviruses are transmitted to human by rodents, resulting in severe acute infections and hemorrhagic fever. Pseudotyped viruses have been widely used as a tool in the study of mammarenaviruses. HIV-1, SIV, FIV-based lentiviral vectors, VSV-based vectors, MLV-based vectors, and reverse genetic approaches have been applied in the construction of pseudotyped mammarenaviruses. Pseudotyped mammarenaviruses are commonly used in receptor research, neutralizing antibody detection, inhibitor screening, viral virulence studies, functional analysis of N-linked glycans, and studies of viral infection, endocytosis, and fusion mechanisms.

Employing an immunoinformatics approach revealed potent multi-epitope based subunit vaccine for lymphocytic choriomeningitis virus.

BACKGROUND: Lymphocytic choriomeningitis virus (LCMV) infects many individuals worldwide and causes severe infection in the immunosuppressant recipient, spontaneous abortion, and congenital disabilities in infants. OBJECTIVES: There is no specific vaccine or therapeutics available to protect against LCMV infection; thus, there is a need to design a potential vaccine to combat the virus by developing immunity in the population. Herein, we attempted to design a potent multi-epitope vaccine for LCMV using immunoinformatics methods. METHODS: The whole proteome of the virus was screened and mapped to extract immunodominant B-cell and T-cell epitopes which were fused with appropriate linkers (EAAAK, GGGS, AAY, GPGPG, and AAY), PADRE sequence (13aa) and an adjuvant (50 S ribosomal protein L7/L12) to formulate a multi-epitope vaccine ensemble. Codon adaptation and in silico cloning of the constructed vaccine were carried out using bioinformatics tools. The secondary and tertiary structure of the vaccine construct was predicted and refined. The physicochemical profile of the designed vaccine was analyzed, and the multi-epitope vaccine's potential to bind Toll-like receptors (TLR2 and TLR4) was evaluated through molecular docking and molecular dynamics simulations. Computational immune simulation of the designed vaccine antigen was performed using the C-ImmSim server. RESULTS: The designed multi-epitope-based vaccine (613 aa) comprised 26 immunodominant (six B-cell, nine cytotoxic T lymphocytes, and 11 helper T lymphocytes) epitopes and is predicted antigenic, non-toxic, non-allergen, soluble, and topographically accessible with a suitable physicochemical profile. The designed vaccine is expected to cover a broad worldwide population (96.35 %) and stimulate a robust adaptive immune response against the virus upon administration. In silico cloning of the constructed vaccine in PET28a (+) vector ensured its optimal expression in the Escherichia coli system. Molecular docking, molecular dynamics simulation, and binding free energy estimation collectively support the stability and energetically favourable interaction of the modeled vaccine-TLR2/4 complexes. CONCLUSION: The designed multi-epitope vaccine in the present study could serve as a potential vaccine candidate to protect against LMCV infection; however, the experimental validation and safety testing of the vaccine is warranted to validate the study's outcomes.

Molecular Engineering of a Mammarenavirus with Unbreachable Attenuation.

Several mammarenaviruses cause severe hemorrhagic fever (HF) disease in humans and pose important public health problems in their regions of endemicity. There are no United States (US) Food and Drug Administration (FDA)-approved mammarenavirus vaccines, and current anti-mammarenavirus therapy is limited to an off-label use of ribavirin that has limited efficacy. Mammarenaviruses are enveloped viruses with a bi-segmented negative-strand RNA genome. Each genome segment contains two open reading frames (ORF) separated by a noncoding intergenic region (IGR). The large (L) segment encodes the RNA dependent RNA polymerase, L protein, and the Z matrix protein, whereas the small (S) segment encodes the surface glycoprotein precursor (GPC) and nucleoprotein (NP). In the present study, we document the generation of a recombinant form of the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) expressing a codon deoptimized (CD) GPC and containing the IGR of the S segment in both the S and L segments (rLCMV/IGR-CD). We show that rLCMV/IGR-CD is fully attenuated in C57BL/6 (B6) mice but able to provide complete protection upon a single administration against a lethal challenge with LCMV. Importantly, rLCMV/IGR-CD exhibited an unbreachable attenuation for its safe implementation as a live-attenuated vaccine (LAV). IMPORTANCE Several mammarenaviruses cause severe disease in humans and pose important public health problems in their regions of endemicity. Currently, no FDA-licensed mammarenavirus vaccines are available, and anti-mammarenaviral therapy is limited to an off-label use of ribavirin whose efficacy is controversial. Here, we describe the generation of recombinant version of the prototypic mammarenavirus lymphocytic choriomeningitis virus (rLCMV) combining the features of a codon deoptimized (CD) GPC and the noncoding intergenic region (IGR) of the S segment in both S and L genome segments, called rLCMV/IGR-CD. We present evidence that rLCMV/IGR-CD has excellent safety and protective efficacy features as live-attenuated vaccine (LAV). Importantly, rLCMV/IGR-CD prevents, in coinfected mice, the generation of LCMV reassortants with increased virulence. Our findings document a well-defined molecular strategy for the generation of mammarenavirus LAV candidates able to trigger long-term protective immunity, upon a single immunization, while exhibiting unique enhanced safety features, including unbreachable attenuation.

Generation of Bi-Reporter-Expressing Tri-Segmented Arenavirus.

Reverse genetics systems provide a powerful tool to generate recombinant arenavirus expressing reporters to facilitate the investigation of the arenavirus life cycle and also for the discovery of antiviral countermeasures. The plasmid-encoded viral ribonucleoprotein components initiate the transcription and replication of a plasmid-driven full-length viral genome, resulting in infectious virus. Thereby, this approach is ideal for the generation of recombinant arenaviruses expressing reporter genes that can be used as valid surrogates for virus replication. By splitting the small viral segment (S) into two viral segments (S1 and S2), each of them encoding a reporter gene, recombinant tri-segmented arenavirus can be rescued. Bi-reporter-expressing recombinant tri-segmented arenaviruses represent an excellent tool to study the biology of arenaviruses, including the identification and characterization of both prophylactic and therapeutic countermeasures for the treatment of arenaviral infections. In this chapter, we describe a detailed protocol on the generation and in vitro characterization of recombinant arenaviruses containing a tri-segment genome expressing two reporter genes based on the prototype member in the family, lymphocytic choriomeningitis virus (LCMV). Similar experimental approaches can be used for the generation of bi-reporter-expressing tri-segment recombinant viruses for other members in the arenavirus family.

Lymphocytic Choriomeningitis Virus Infection, Australia.

During a mouse plague in early 2021, a farmer from New South Wales, Australia, sought treatment for aseptic meningitis and was subsequently diagnosed with locally acquired lymphocytic choriomeningitis virus infection. Whole-genome sequencing identified a divergent and geographically distinct lymphocytic choriomeningitis virus strain compared with other published sequences.

Replication-Deficient Lymphocytic Choriomeningitis Virus-Vectored Vaccine Candidate for the Induction of T Cell Immunity against Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) represents a major burden to global health, and refined vaccines are needed. Replication-deficient lymphocytic choriomeningitis virus (rLCMV)-based vaccine vectors against cytomegalovirus have proven safe for human use and elicited robust T cell responses in a large proportion of vaccine recipients. Here, we developed an rLCMV vaccine expressing the Mtb antigens TB10.4 and Ag85B. In mice, rLCMV elicited high frequencies of polyfunctional Mtb-specific CD8 and CD4 T cell responses. CD8 but not CD4 T cells were efficiently boosted upon vector re-vaccination. High-frequency responses were also observed in neonatally vaccinated mice, and co-administration of rLCMV with Expanded Program of Immunization (EPI) vaccines did not result in substantial reciprocal interference. Importantly, rLCMV immunization significantly reduced the lung Mtb burden upon aerosol challenge, resulting in improved lung ventilation. Protection was associated with increased CD8 T cell recruitment but reduced CD4 T cell infiltration upon Mtb challenge. When combining rLCMV with BCG vaccination in a heterologous prime-boost regimen, responses to the rLCMV-encoded Mtb antigens were further augmented, but protection was not significantly different from rLCMV or BCG vaccination alone. This work suggests that rLCMV may show utility for neonatal and/or adult vaccination efforts against pulmonary tuberculosis.

Arenaviral infection causes bleeding in mice due to reduced serotonin release from platelets.

Bleeding correlates with disease severity in viral hemorrhagic fevers. We found that the increase in type I interferon (IFN-I) in mice caused by infection with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV; an arenavirus) reduced the megakaryocytic expression of genes encoding enzymes involved in lipid biosynthesis (cyclooxygenase 1 and thromboxane A synthase 1) and a thrombopoietic transcription factor (Nf-e2). The decreased expression of these genes was associated with reduced numbers of circulating platelets and defects in the arachidonic acid synthetic pathway, thereby suppressing serotonin release from δ-granules in platelets. Bleeding resulted when severe thrombocytopenia and altered platelet function reduced the amount of platelet-derived serotonin below a critical threshold. Bleeding was facilitated by the absence of the activity of the kinase Lyn or the administration of aspirin, an inhibitor of arachidonic acid synthesis. Mouse platelets were not directly affected by IFN-I because they lack the receptor for the cytokine (IFNAR1), suggesting that transfusion of normal platelets into LCMV-infected mice could increase the amount of platelet-released serotonin and help to control hemorrhage.

Single MHC-I Expression Promotes Virus-Induced Liver Immunopathology.

Major histocompatibility complex I (MHC-I) molecules present epitopes on the cellular surface of antigen-presenting cells to prime cytotoxic clusters of differentiation 8 (CD8)+ T cells (CTLs), which then identify and eliminate other cells such as virus-infected cells bearing the antigen. Human hepatitis virus cohort studies have previously identified MHC-I molecules as promising predictors of viral clearance. However, the underlying functional significance of these predictions is not fully understood. Here, we show that expression of single MHC-I isomers promotes virus-induced liver immunopathology. Specifically, using the lymphocytic choriomeningitis virus (LCMV) model system, we found MHC-I proteins to be highly up-regulated during infection. Deletion of one of the two MHC-I isomers histocompatibility antigen 2 (H2)-Db or H2-Kb in C57Bl/6 mice resulted in CTL activation recognizing the remaining MHC-I with LCMV epitopes in increased paucity. This increased CTL response resulted in hepatocyte death, increased caspase activation, and severe metabolic changes in liver tissue following infection with LCMV. Moreover, depletion of CTLs abolished LCMV-induced pathology in these mice with resulting viral persistence. In turn, natural killer (NK) cell depletion further increased antiviral CTL immunity and clearance of LCMV even in the presence of a single MHC-I isomer. Conclusion: Our results suggest that uniform MHC-I molecule expression promotes enhanced CTL immunity during viral infection and contributes to increased CTL-mediated liver cell damage that was alleviated by CD8 or NK cell depletion.

A small stem-loop-forming region within the 3'-UTR of a nonpolyadenylated LCMV mRNA promotes translation.

Mammalian arenavirus (mammarenavirus) mRNAs are characterized by 5'-capped and 3'-nonpolyadenylated untranslated regions (UTRs). We previously reported that the nonpolyadenylated 3'-UTR of viral mRNA (vmRNA), which is derived from the noncoding intergenic region (IGR), regulates viral protein levels at the posttranscriptional level. This finding provided the basis for the development of novel live-attenuated vaccines (LAVs) against human pathogenic mammarenaviruses. Detailed information about the roles of specific vmRNA 3'-UTR sequences in controlling translation efficiency will help in understanding the mechanism underlying attenuation by IGR manipulations. Here, we characterize the roles of cis-acting mRNA regulatory sequences of a prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), in modulating translational efficiency. Using in vitro transcribed RNA mimics encoding a reporter gene, we demonstrate that the 3'-UTR of nucleoprotein (NP) mRNA without a poly(A) tail promotes translation in a poly(A)-binding protein-independent manner. Comparison with the 3'-UTR of glycoprotein precursor mRNA, which is translated less efficiently, revealed that a 10-nucleotide sequence proximal to the NP open reading frame is essential for promoting translation. Modification of this 10-nucleotide sequence also impacted reporter gene expression in recombinant LCMV. Our findings will enable rational design of the 10-nucleotide sequence to further improve our mammarenavirus LAV candidates and to develop a novel LCMV vector capable of controlling foreign gene expression.

Progranulin mediates immune evasion of pancreatic ductal adenocarcinoma through regulation of MHCI expression.

Immune evasion is indispensable for cancer initiation and progression, although its underlying mechanisms in pancreatic ductal adenocarcinoma (PDAC) are not fully known. Here, we characterize the function of tumor-derived PGRN in promoting immune evasion in primary PDAC. Tumor- but not macrophage-derived PGRN is associated with poor overall survival in PDAC. Multiplex immunohistochemistry shows low MHC class I (MHCI) expression and lack of CD8+ T cell infiltration in PGRN-high tumors. Inhibition of PGRN abrogates autophagy-dependent MHCI degradation and restores MHCI expression on PDAC cells. Antibody-based blockade of PGRN in a PDAC mouse model remarkably decelerates tumor initiation and progression. Notably, tumors expressing LCMV-gp33 as a model antigen are sensitized to gp33-TCR transgenic T cell-mediated cytotoxicity upon PGRN blockade. Overall, our study shows a crucial function of tumor-derived PGRN in regulating immunogenicity of primary PDAC.

A Randomized Dose-Escalating Phase I Trial of a Replication-Deficient Lymphocytic Choriomeningitis Virus Vector-Based Vaccine Against Human Cytomegalovirus.

BACKGROUND: A vaccine (HB-101) consisting of 2 nonreplicating lymphocytic choriomeningitis virus (LCMV) vectors expressing the human cytomegalovirus antigens glycoprotein B (gB) and the 65-kD phosphoprotein (pp65), respectively, is in development to prevent cytomegalovirus infection. METHODS: HB-101 was tested in cytomegalovirus-naive, healthy adults in a randomized, double-blind, placebo-controlled, dose-escalation Phase I trial. Fifty-four subjects received low, medium, or high dose of HB-101 or placebo by intramuscular administration at Month 0, 1, and 3. Safety and immunogenicity were the respective primary and secondary endpoints. Subjects were followed for 12 months after the initial immunization. RESULTS: Vaccination was associated with transient mild to moderate adverse events. HB-101 administration induced dose-dependent gB- and pp65-specific cellular responses, dominated by pp65-specific CD8 T cells, a high fraction of which were polyfunctional. Two administrations were sufficient to elicit dose-dependent gB-binding and cytomegalovirus-neutralizing antibodies (Abs). Cytomegalovirus-specific immune responses were boosted after each administration. Only 1 of 42 vaccine recipients mounted a transient LCMV vector-neutralizing Ab response. CONCLUSIONS: HB-101 was well tolerated and induced cytomegalovirus-specific polyfunctional CD8 T-cell and neutralizing Ab responses in the majority of subjects. Lack of vector-neutralizing Ab responses should facilitate booster vaccinations. These results justify further clinical evaluation of this vaccine candidate.

Development of a high-throughput RT-PCR based viral infectivity assay for monitoring the stability of a replicating recombinant Lymphocytic Choriomeningitis viral vector.

Traditional virus infectivity titration methods for lymphocytic choriomeningitis virus (LCMV) are laborious, time-consuming, and low-throughput (e.g., focus forming unit (FFA) assay). In this report, we developed a high-throughput reverse transcription quantitative PCR (RT-qPCR)-based virus infectivity assay for relative quantitation of a live, recombinant replicating LCMV -based viral vector (TT1). This in vitro infectivity assay demonstrated a 4-log linear range for TT1 titer quantitation. A high positive Pearson correlation coefficient value (≥ 0.80) was obtained between the RT-qPCR vs. the "gold-standard" FFU assay when comparing the stability profiles of stressed TT1 vector samples. In addition to the RT-qPCR infectivity assay, the stability of the TT1 vector upon freeze-thaw stress was investigated further with complementary viral particle characterization techniques (e.g., TEM, NTA, MFI). Correlations between viral infectivity and particle measurements during forced degradation studies were observed to be specific to the TT1 vector and its various formulations and such results facilitated the rank-ordering of formulation conditions. Overall, this infectivity RT-qPCR method showed increased sample throughput and improved assay flexibility compared to traditional viral infectivity assays. These results are discussed in the context of enabling future TT1 vector formulation development work, and potential utilization as an in-process monitoring tool during TT1 vector manufacturing.

New Perspective on the Geographic Distribution and Evolution of Lymphocytic Choriomeningitis Virus, Central Europe.

Lymphocytic choriomeningitis virus (LCMV) is an Old World mammarenavirus found worldwide because of its association with the house mouse. When LCMV spills over to immunocompetent humans, the virus can cause aseptic meningitis; in immunocompromised persons, systemic infection and death can occur. Central Europe is a strategic location for the study of LCMV evolutionary history and host specificity because of the presence of a hybrid zone (genetic barrier) between 2 house mouse subspecies, Mus musculus musculus and M. musculus domesticus. We report LCMV prevalence in natural mouse populations from a Czech Republic-Germany transect and genomic characterization of 2 new LCMV variants from the Czech Republic. We demonstrate that the main division in the LCMV phylogenetic tree corresponds to mouse host subspecies and, when the virus is found in human hosts, the mouse subspecies found at the spillover location. Therefore, LCMV strains infecting humans can be predicted by the genetic structure of house mice.