Enhanced Virus Clearance by Early Inducible Lymphocytic Choriomeningitis Virus-Neutralizing Antibodies in Immunoglobulin-Transgenic Mice

Following infection of mice with lymphocytic choriomeningitis virus (LCMV), virus-neutralizing antibodies appear late, after 30 to 60 days. Such neutralizing antibodies play an important role in protection against reinfection. To analyze whether a neutralizing antibody response which developed earlier could contribute to LCMV clearance during the acute phase of infection, we generated transgenic mice expressing LCMV-neutralizing antibodies. Transgenic mice expressing the immunoglobulin μ heavy chain of the LCMV-neutralizing monoclonal antibody KL25 (H25 transgenic mice) mounted LCMV-neutralizing immunoglobulin M (IgM) serum titers within 8 days after infection. This early inducible LCMV-neutralizing antibody response significantly improved the host’s capacity to clear the infection and did not cause an enhancement of disease after intracerebral (i.c.) LCMV infection. In contrast, mice which had been passively administered LCMV-neutralizing antibodies and transgenic mice exhibiting spontaneous LCMV-neutralizing IgM serum titers (HL25 transgenic mice expressing the immunoglobulin μ heavy and the κ light chain) showed an enhancement of disease after i.c. LCMV infection. Thus, early-inducible LCMV-neutralizing antibodies can contribute to viral clearance in the acute phase of the infection and do not cause antibody-dependent enhancement of disease.Against many cytopathic viruses such as poliovirus, influenza virus, rabies virus, and vesicular stomatitis virus, protective virus-neutralizing antibodies are generated early, within 1 week after infection (3, 31, 36, 44, 49). In contrast, several noncytopathic viruses (e.g., human immunodeficiency virus and hepatitis viruses B and C in humans or lymphocytic choriomeningitis virus [LCMV] in mice) elicit poor and delayed virus-neutralizing antibody responses (1, 7, 20, 24, 27, 35, 45, 48).In the mouse, the natural host of LCMV, the acute LCMV infection is predominantly controlled by cytotoxic T lymphocytes (CTLs) in an obligatory perforin-dependent manner (13, 18, 28, 50). In addition to the CTL response, LCMV-specific antibodies are generated. Early after infection (by day 8), a strong antibody response specific for the internal viral nucleoprotein (NP) is mounted (7, 19, 23, 28). These early LCMV NP-specific antibodies exhibit no virus-neutralizing capacity (7, 10). Results from studies of B-cell-depleted mice and B-cell-deficient mice implied that the early LCMV NP-specific antibodies are not involved in the clearance of LCMV (8, 11, 12, 40). Late after infection (between days 30 and day 60), LCMV-neutralizing antibodies develop (7, 19, 22, 28, 33); these antibodies are directed against the surface glycoprotein (GP) of LCMV (9, 10). LCMV-neutralizing antibodies have an important function in protection against reinfection (4, 6, 38, 41, 47).In some viral infections, subprotective virus-neutralizing antibody titers can enhance disease rather than promote host recovery (i.e., exhibit antibody-dependent enhancement of disease [ADE] [14, 15, 21, 46]). For example, neutralizing antibodies are involved in the resolution of a primary dengue virus infection and in the protection against reinfection. However, if subprotective neutralizing antibody titers are present at the time of reinfection, a severe form of the disease (dengue hemorrhagic fever/dengue shock syndrome [15, 21]), which might be caused by Fc receptor-mediated uptake of virus-antibody complexes leading to an enhanced infection of monocytes (15, 16, 25, 39), can develop. Similarly, an enhancement of disease after intracerebral (i.c.) LCMV infection was observed in mice which had been treated with virus-neutralizing antibodies before the virus challenge (6). ADE in LCMV-infected mice was either due to an enhanced infection of monocytes by Fc receptor-mediated uptake of antibody-virus complexes or due to CTL-mediated immunopathology caused by an imbalanced virus spread and CTL response.To analyze whether LCMV-neutralizing antibodies generated early after infection improve the host’s capacity to clear the virus or enhance immunopathological disease, immunoglobulin (Ig)-transgenic mice expressing LCMV-neutralizing IgM antibodies were generated. After LCMV infection of transgenic mice expressing the Ig heavy chain (H25 transgenic mice), LCMV-neutralizing serum antibodies were mounted within 8 days, which significantly improved the host’s capacity to eliminate LCMV. H25 transgenic mice did not show any signs of ADE after i.c. LCMV infection.Transgenic mice expressing the Ig heavy and light chains (HL25 transgenic mice) exhibited spontaneous LCMV-neutralizing serum antibodies and confirmed the protective role of preexisting LCMV-neutralizing antibodies, even though the neutralizing serum antibodies were of the IgM isotype. Similar to mice which had been treated with LCMV-neutralizing antibodies, HL25 transgenic mice developed an enhanced disease after i.c. LCMV infection, which indicated that ADE was due to an imbalance between virus spread and CTL response. Thus, the early-inducible LCMV-neutralizing antibody response significantly enhanced clearance of the acute infection without any risk of causing ADE.     

Plaque Size Heterogeneity: a Genetic Trait of Lymphocytic Choriomeningitis Virus

All of the ten strains of lymphocytic choriomeningitis virus assayed on BHK 21/13S cells showed various degrees of plaque size heterogeneity. The amount of virus released from these plaques was usually very small because of rapid photodynamic inactivation by neutral red. When virus from large and small plaques of a specific strain was plated, the same distribution of plaque size was obtained from each clone. Although it was shown that surface virus could possibly be randomly distributed at the time of addition of neutral red overlays, no virus could be isolated from nonplaque areas. Two different strains of virus (CA1371 and WE) with markedly different plaque size ranges were separated by plaque excision from plates infected with a mixture of both viruses.     

Seroepidemiological Survey of Lymphocytic Choriomeningitis Virus in Wild House Mice in China with Particular Reference to Their Subspecies

Serum samples from 337 wild house mice (Mus musculus) from 35 sites in China, collected in 1992 and 1993, were examined for antibodies against lymphocytic choriomeningitis virus (LCMV). Ten samples from eight sites were found to contain such antibodies. Six of the eight positive sites were located in the territory of M. m. gansuensis. One of the other two sites was located in the territory of M. m. castaneus in southern China and the other site was in a habitat of M. m. castaneus which had invaded into the western end of the territory of M. m. homourus. It seems likely that LCMV is distributed in the territories of M. m. gansuensis and M. m. castaneus in China. This is the first report of detection of these antibodies in wild house mice in China and specifically in the territories of M. m. gansuensis and M. m. castaneus.     

Biophysical and Biochemical Characterization of Lymphocytic Choriomeningitis Virus: IV. Strain Differences

Biological, biochemical, and biophysical properties of three lymphocytic choriomeningitis (LCM) virus strains were compared. The biological property examined was the concentration range of virus which would, when injected into neonates, cause a carrier state. The dosage range for the CA1371 and Traub strains was found to be as broad as the limits examined (5 to 100 ld(50) units/mouse). The WCP strain, however, would only produce carriers within a 3 to 5 ld(50) range. The biochemical properties examined were the growth rates in tissue culture and the effect of varying the input ratio of virus to cells. With identical input ratios, the Traub strain reached a peak titer 32 hr after infection. The CA1371 and WCP strain reached their peaks at the 40th hr. With a 10-fold decrease in the amount of CA1371 virus per cell, peak titer (as high as in the above experiments) was not obtained until 56 hr postinfection. The biophysical properties examined were stability in density gradients and inactivation rates at 4C. In potassium tartrate gradients, full recovery of the CA1371 and WCP strain could be achieved. However, inactivation kinetics showed that only the CA1371 strain was much more stable than the Traub-LCM. The realization that marked differences in LCM strains exist is discussed in relation to certain inconsistencies in the literature.     

Effect of Serum on the Yield of Lymphocytic Choriomeningitis Virus in Baby Hamster Kidney Cells

The yields of the Armstrong and WE strains of lymphocytic choriomeningitis virus in baby hamster kidney (BHK) cells cultivated in either bovine, calf, fetal bovine, or horse serum were investigated. Lines of BHK cells were established in these sera. When the infected cell lines were observed by immunofluorescence, the per cent fluorescing cells for a given virus strain did not vary. However, for both strains, the extracellular virus yields per cell were significantly greater in the fetal bovine-cell line than in the other serum-cell lines.     

Role of Interferon Regulatory Factor 7 in T Cell Responses during Acute Lymphocytic Choriomeningitis Virus Infection

Type I interferons (IFNs), predominantly IFN-α and -β, play critical roles in both innate and adaptive immune responses against viral infections. Interferon regulatory factor 7 (IRF7), a key innate immune molecule in the type I IFN signaling pathway, is essential for the type I IFN response to many viruses, including lymphocytic choriomeningitis virus (LCMV). Here, we show that although IRF7 knockout (KO) mice failed to control the replication of LCMV in the early stages of infection, they were capable of clearing LCMV infection. Despite the lack of type I IFN production, IRF7 KO mice generated normal CD4⁺ T cell responses, and the expansion of naïve CD8⁺ T cells into primary CD8⁺ T cells specific for LCMV GP_33–41 was relatively normal. In contrast, the expansion of the LCMV NP₃₉₆-specific CD8⁺ T cells was severely impaired in IRF7 KO mice. We demonstrated that this defective CD8⁺ T cell response is due neither to an impaired antigen-presenting system nor to any intrinsic role of IRF7 in CD8⁺ T cells. The lack of a type I IFN response in IRF7 KO mice did not affect the formation of memory CD8⁺ T cells. Thus, the present study provides new insight into the impact of the innate immune system on viral pathogenesis and demonstrates the critical contribution of innate immunity in controlling virus replication in the early stages of infection, which may shape the quality of CD8⁺ T cell responses.     

The Bacteriostatic Protein Lipocalin 2 Is Induced in the Central Nervous System of Mice with West Nile Virus Encephalitis

Lipocalin 2 (Lcn2) is a bacteriostatic factor produced during the innate immune response to bacterial infection. Whether Lcn2 has a function in viral infection is unknown. We investigated the regulation and function of Lcn2 in the central nervous system (CNS) of mice during West Nile virus (WNV) encephalitis. Lcn2 mRNA and protein were induced in the brain by day 5, and this induction increased further by day 7 postinfection but was delayed compared with the induction of the toll-like receptor 3 (TLR3) gene, retinoic acid-inducible gene 1 (RIG-I), and melanoma differentiation-associated protein 5 (MDA5) gene. The Lcn2 mRNA and protein were both found at high levels in the choroid plexus, vascular endothelium, macrophage/microglia, and astrocytes. However, some neuronal subsets contained Lcn2 protein but no detectable mRNA. In Lcn2 knockout (KO) mice, with the exception of CXC motif chemokine 5 (CXCL5), which was significantly more downregulated than in wild-type (WT) mice, expression levels of a number of other host response genes were similar in the two genotypes. The brain from Lcn2 and WT mice with WNV encephalitis contained similar numbers of infiltrating macrophages, granulocytes, and T cells. Lcn2 KO and WT mice had no significant difference in tissue viral loads or survival after infection with different doses of WNV. We conclude that Lcn2 gene expression is induced to high levels in a time-dependent fashion in a variety of cells and regions of the CNS of mice with WNV encephalitis. The function of Lcn2 in the host response to WNV infection remains largely unknown, but our data indicate that it is dispensable as an antiviral or immunoregulatory factor in WNV encephalitis.     

Cross-species transmission of simian foamy virus to humans in rural Gabon, Central Africa

In order to characterize simian foamy retroviruses (SFVs) in wild-born nonhuman primates (NHPs) in Gabon and to investigate cross-species transmission to humans, we obtained 497 NHP samples, composed of 286 blood and 211 tissue (bush meat) samples. Anti-SFV antibodies were found in 31 of 286 plasma samples (10.5%). The integrase gene sequence was found in 38/497 samples, including both blood and tissue samples, with novel SFVs in several Cercopithecus species. Of the 78 humans, mostly hunters, who had been bitten or scratched by NHPs, 19 were SFV seropositive, with 15 cases confirmed by PCR. All but one were infected with ape SFV. We thus found novel SFV strains in NHPs in Gabon and high cross-species transmission of SFVs from gorilla bites.     

PC61 (Anti-CD25) Treatment Inhibits Influenza A Virus-Expanded Regulatory T Cells and Severe Lung Pathology during a Subsequent Heterologous Lymphocytic Choriomeningitis Virus Infection

Prior immunity to influenza A virus (IAV) in mice changes the outcome to a subsequent lymphocytic choriomeningitis virus (LCMV) infection and can result in severe lung pathology, similar to that observed in patients that died of the 1918 H1N1 pandemic. This pathology is induced by IAV-specific memory CD8⁺ T cells cross-reactive with LCMV. Here, we discovered that IAV-immune mice have enhanced CD4⁺ Foxp3⁺ T-regulatory (Treg) cells in their lungs, leading us to question whether a modulation in the normal balance of Treg and effector T-cell responses also contributes to enhancing lung pathology upon LCMV infection of IAV-immune mice. Treg cell and interleukin-10 (IL-10) levels remained elevated in the lungs and mediastinal lymph nodes (mLNs) throughout the acute LCMV response of IAV-immune mice. PC61 treatment, used to decrease Treg cell levels, did not change LCMV titers but resulted in a surprising decrease in lung pathology upon LCMV infection in IAV-immune but not in naive mice. Associated with this decrease in pathology was a retention of Treg in the mLN and an unexpected partial clonal exhaustion of LCMV-specific CD8⁺ T-cell responses only in IAV-immune mice. PC61 treatment did not affect cross-reactive memory CD8⁺ T-cell proliferation. These results suggest that in the absence of IAV-expanded Treg cells and in the presence of cross-reactive memory, the LCMV-specific response was overstimulated and became partially exhausted, resulting in a decreased effector response. These studies suggest that Treg cells generated during past infections can influence the characteristics of effector T-cell responses and immunopathology during subsequent heterologous infections. Thus, in humans with complex infection histories, PC61 treatment may lead to unexpected results.     

Usefulness of a Fourth Generation ELISA Assay for the Reliable Identification of HCV Infection in HIV-Positive Adults from Gabon (Central Africa)

Background/Objectives

Guidelines for optimized HCV screening are urgently required in Africa, especially for patients infected with HIV, who sometimes show false positive or false negative reactivity in anti-HCV antibody assays. Here, we assessed the usefulness of a fourth-generation HCV Ag-Ab ELISA for the identification of active HCV infection in HIV-positive patients.

Methods

This cross-sectional study was conducted between 03/2010 and 01/2013 and included 762 Gabonese HIV-positive adult patients. The results of ELISA (Monolisa HCV Ag-Ab ULTRA, Bio-Rad) were compared with those obtained by RT-PCR (gold standard). The optimal ELISA signal-to-cutoff (S/CO) ratio to identify patients with active hepatitis C (positive HCV RNA) was determined. Specimens were further tested by the INNO-LIA HCV Score assay (Innogenetics) and the Architect HCV Ag kit (Abbott) to define the best diagnostic strategy.

Results

Sixty-seven patients tested positive for HCV (S/CO ratio ≥ 1) by ELISA. Of these, 47 (70.1%) tested positive for HCV RNA. The optimal S/CO associated with active HCV infection was 1.7. At this threshold, the sensitivity of ELISA was 97.9% (95% confidence interval (CI) 90.0–99.9%), its specificity was 91.3% (95% CI 85.0–95.5%), and HCV seroprevalence rate was 7.3% (56/762) (95% CI 5.6–9.4%). Among 57 HCV-seropositive patients with available INNO-LIA results, false reactivity was identified in 14 (24.6%), resolved HCV infection in two (3.5%), possible acute HCV infections in nine (15.8%) and likely chronic HCV infections in 32 (56.1%) patients. HCV core Ag was undetectable in 14/15 (93.3%) specimens that tested negative for HCV RNA whereas it was quantified in 34 (out of 39, 87.2%) samples that tested positive for HCV RNA.

Conclusions

Our study provides comprehensive guidance for HCV testing in Gabon, and will help greatly clinicians to improve case definitions for both the notification and surveillance of HCV in patients co-infected with HIV.     

Microbotanical Evidence of Domestic Cereals in Africa 7000 Years Ago

The study of plant exploitation and early use of cereals in Africa has seen over the years a great input from charred and desiccated macrobotanical remains. This paper presents the results of one of the few examples in Africa of microbotanical analyses. Three grave contexts of phytolith-rich deposits and the dental calculus of 20 individuals were analysed from two Neolithic cemeteries in North and Central Sudan. The radiocarbon-dated phytoliths from the burial samples show the presence of Near East domestic cereals in Northern Sudan at least 7000 years ago. Phytoliths also indicate the exploitation of wild, savannah-adapted millets in Central Sudan between 7500 and 6500 years ago. The calculus samples contained starch grains from wheat/barley, pulses and millets, as well as panicoid phytoliths. This evidence shows that Near East domestic cereals were consumed in Northern Africa at least 500 years earlier than previously thought.     

Modern pollen rain in savanna and forest ecosystems of Gabon and Cameroon,Central Atlantic Africa

Eighty modern soil surface and litter samples from southern Cameroon and Gabon, Central Atlantic Africa (5°N–4°S, 10°–15°W), were analysed for pollen content. The samples are distributed among two main vegetation types: savanna (8 samples) and forest (71 samples). The aim of this study is to provide new data on the modern pollen rain in the Guineo-Congolian phytogeographical region, mainly in forest communities (secondary and mature forests on well drained soils, and hygrophilous forests) and to interpret these data using diagrams of pollen percentages and numerical analyses. The savannas are well identified by high frequencies of non-arboreal pollen with as pollen marker the Poaceae, and the forests by high frequencies of arboreal pollen with as important families the Burseraceae, Caesalpiniaceae, Mimosaceae, Euphorbiaceae and Sapindaceae. Within the forest ecosystem, secondary and mature forests on well drained soils can be differentiated on the basis of distinct assemblages of tree pollen taxa such as Zanthoxylum, Phyllanthus, Tetrorchidium, Margaritaria discoidea in secondary forest spectra and abundance of Burseraceae, Caesalpiniaceae, Sapindaceae in mature forest ones. In addition, hygrophilous forests are well identified by the presence of high pollen contributors such as Uapaca, Nauclea, Macaranga and Raphia. This work shows that the major vegetation communities occurring today in Cameroon and Gabon can be well differentiated by their pollen assemblages.     

Altered Tryptophan Metabolism in Mice with Herpes Simplex Virus Encephalitis: Increases in Spinal Cord Quinolinic Acid

Mice infected with the herpes simplex virus, type-1, developed a paralysis which was associated with increased levels of the neurotoxin quinolinic acid (QUIN). The largest increases in QUIN were observed in the spinal cord with much smaller changes in the rostral forebrain or serum. The time course for the paralysis coincided with the increase in spinal cord QUIN, a maximal 40-fold elevation, at 7–10 days post infection. The time course suggested that the increases in QUIN were due to its local synthesis. Consistent with this possibility, herpes virus-infected mice had increased activities of indoleamine 2,3-dioxygenase and kynurenine hydroxylase (two key enzymes in QUIN formation), when compared to non-infected controls. Since QUIN is formed by activated macro-phages, these new data are consistent with QUIN formation as part of the host response to a pathogen whose importance is discussed.     

Isolation of Two Plaque Mutants of Western Equine Encephalitis Virus Differing in Virulence for Mice

Two plaque mutants were isolated from tissue cultures infected persistently with Western equine encephalitis virus. A large plaque mutant proved to be markedly avirulent for mice.