Squalene-containing licensed adjuvants enhance strain-specific antibody responses against the influenza hemagglutinin and induce subtype-specific antibodies against the neuraminidase.

While seasonal influenza vaccines are usually non-adjuvanted, H1N1pdm09 vaccines were formulated with different squalene-containing adjuvants, to enable the reduction of antigen content thus increasing the number of doses available. To comparatively assess the effects of these adjuvants on antibody responses against matched and mismatched strains, and to correlate antibody levels with protection from disease, ferrets were immunized with 2µg of commercial H1N1pdm09 vaccine antigen alone or formulated with different licensed adjuvants. The use of squalene-containing adjuvants increased neutralizing antibody responses around 100-fold, and resulted in a significantly reduced viral load after challenge with a matched strain. While all animals mounted strong total antibody responses against the homologous H1N1 hemagglutinin (HA) protein, which correlated with the respective neutralizing antibody titers, no reactivity with the divergent H3, H5, H7, and H9 proteins were detected. Only the adjuvanted vaccines also induced antibodies against the neuraminidase (NA) protein, which were able to also recognize NA proteins from other N1 carrying strains. These findings not only support the use of squalene-containing adjuvants in dose-sparing strategies but also support speculations that the induction of NA-specific responses associated with the use of these adjuvants may confer partial protection to heterologous strains carrying the same NA subtype.

Foodborne-Transmitted Prions From the Brain of Cows With Bovine Spongiform Encephalopathy Ascend in Afferent Neurons to the Simian Central Nervous System and Spread to Tonsils and Spleen at a Late Stage of the Incubation Period.

BACKGROUND: Protease-resistant prion protein (PrP(res)) accumulation in lymphoreticular tissues indicates prion infection. To date, tonsillectomy and appendectomy samples have been used in population prevalence surveys to detect clinically silent carriers of variant Creutzfeldt-Jakob disease (vCJD). However, the temporal sequence of prion spread in the human body is still not known. We therefore traced the temporal-spatial pattern of PrP(res) accumulation in the body of a simian vCJD model. METHODS: Cynomolgus monkeys were fed brain of (eleven) cows with bovine spongiform encephalopathy, and some were euthanized before and some after onset of neurological signs. PrP(res) was detected in tissues by a paraffin-embedded tissue blot technique and a semiquantitative Western immunoblot assay. RESULTS: Bovine spongiform encephalopathy (BSE)-associated prions were preferentially transported from the gut to the central nervous system (CNS) along sensory nerve fibers and initially entered the simian CNS at lumbar spinal cord levels. In asymptomatic animals, we found BSE in 50% and 12% of gut- and tonsil-derived samples, respectively. CONCLUSIONS: Unlike in rodents and ruminants, foodborne BSE-associated prions entered the simian CNS via afferent neurons. From sites of initial CNS invasion, prions spread centrifugally to tonsils and spleen at an advanced stage of the incubation period, thus explaining why tonsil specimens were not reliable for detection of simian disease carriers before onset of clinical signs.

Foodborne transmission of bovine spongiform encephalopathy to non-human primates results in preclinical rapid-onset obesity.

Obesity has become one of the largest public health challenges worldwide. Recently, certain bacterial and viral pathogens have been implicated in the pathogenesis of obesity. In the present study, we retrospectively analyzed clinical data, plasma samples and post-mortem tissue specimens derived from a risk assessment study in bovine spongiform encephalopathy (BSE)-infected female cynomolgus monkeys (Macaca fascicularis). The original study design aimed to determine minimal infectious doses after oral or intracerebral (i.c.) infection of macaques to assess the risk for humans. High-dose exposures resulted in 100% attack rates and a median incubation time of 4.7 years as described previously. Retrospective analyses of clinical data from high-dosed macaques revealed that foodborne BSE transmission caused rapid weight gain within 1.5 years post infection (ß = 0.915; P<0.0001) which was not seen in age- and sex-matched control animals or i.c. infected animals. The rapid-onset obesity was not associated with impaired pancreatic islet function or glucose metabolism. In the early preclinical phase of oral transmission associated with body weight gain, prion accumulation was confined to the gastrointestinal tract. Intriguingly, immunohistochemical findings suggest that foodborne BSE transmission has a pathophysiological impact on gut endocrine cells which may explain rapid weight gain. To our knowledge, this is the first experimental model which clearly demonstrates that foodborne pathogens can induce obesity.

Foodborne transmission of bovine spongiform encephalopathy to nonhuman primates.

Risk for human exposure to bovine spongiform encephalopathy (BSE)-inducing agent was estimated in a nonhuman primate model. To determine attack rates, incubation times, and molecular signatures, we orally exposed 18 macaques to 1 high dose of brain material from cattle with BSE. Several macaques were euthanized at regular intervals starting at 1 year postinoculation, and others were observed until clinical signs developed. Among those who received ≥5 g BSE-inducing agent, attack rates were 100% and prions could be detected in peripheral tissues from 1 year postinoculation onward. The overall median incubation time was 4.6 years (3.7-5.3). However, for 3 macaques orally exposed on multiple occasions, incubation periods were at least 7-10 years. Before clinical signs were noted, we detected a non-type 2B signature, indicating the existence of atypical prion protein during the incubation period. This finding could affect diagnosis of variant Creutzfeldt-Jakob disease in humans and might be relevant for retrospective studies of positive tonsillectomy or appendectomy specimens because time of infection is unknown.

Dominance of highly divergent feline leukemia virus A progeny variants in a cat with recurrent viremia and fatal lymphoma.

BACKGROUND: In a cat that had ostensibly recovered from feline leukemia virus (FeLV) infection, we observed the reappearance of the virus and the development of fatal lymphoma 8.5 years after the initial experimental exposure to FeLV-A/Glasgow-1. The goals of the present study were to investigate this FeLV reoccurrence and molecularly characterize the progeny viruses. RESULTS: The FeLV reoccurrence was detected by the presence of FeLV antigen and RNA in the blood and saliva. The cat was feline immunodeficiency virus positive and showed CD4+ T-cell depletion, severe leukopenia, anemia and a multicentric monoclonal B-cell lymphoma. FeLV-A, but not -B or -C, was detectable. Sequencing of the envelope gene revealed three FeLV variants that were highly divergent from the virus that was originally inoculated (89-91% identity to FeLV-A/Glasgow-1). In the long terminal repeat 31 point mutations, some previously described in cats with lymphomas, were detected. The FeLV variant tissue provirus and viral RNA loads were significantly higher than the FeLV-A/Glasgow-1 loads. Moreover, the variant loads were significantly higher in lymphoma positive compared to lymphoma negative tissues. An increase in the variant provirus blood load was observed at the time of FeLV reoccurrence. CONCLUSIONS: Our results demonstrate that ostensibly recovered FeLV provirus-positive cats may act as a source of infection following FeLV reactivation. The virus variants that had largely replaced the inoculation strain had unusually heavily mutated envelopes. The mutations may have led to increased viral fitness and/or changed the mutagenic characteristics of the virus.

Inducible expression of chimpanzee prion protein (PrP) in murine PrP knock-out cells.

In transmissible spongiform encephalopathy (TSE) pathogenesis the cellular prion protein (PrP(C)) is converted into its pathogenic PrP(Sc) isoform. Prion protein gene (Prnp) deficient mice (PrP(0/0)) are resistant to PrP(Sc) infection, but following reconstitution of Prnp they regain their susceptibility to infection. Therefore, it is challenging to simulate this natural situation in a cell culture model. We have previously reported the inducible stable expression of a human PrP(C) in murine 3T3 cells. In this study, we used murine PrP(0/0) cells stably expressing exemplarily the chimpanzee Prnp under the control of inducible tetracycline (Tet) system. The Prnp was integrated using a lentiviral vector. Its expression in the engineered PrP(0/0)Chimp1/Tet-Off cell line was analyzed by Western blot (Wb) and fluorescence activated cell sorting (FACS) analyses. PrP(C) was partially purified by using immobilized metal affinity chromatography (IMAC). Compared to all the other cell systems which possess an endogenous PrP(C) expression, here described cell line contains only an overexpressing species specific PrP(C) expression which is tightly regulated and can be turned-off at any time without showing any endogenous host PrP(C) expression. Consequently, a contamination of the isolated PrP(C) is impossible. This cell line potentially offers a new tool for simulation of mice bioassays widely used in TSE infection studies.

Increase in CD230 (cellular prion protein) fluorescence on blood lymphocytes in bovine spongiform encephalopathy-infected nonhuman primates.

BACKGROUND: The cellular prion protein (PrP(c)) plays a central role in prion diseases such as variant Creutzfeldt-Jakob disease. This disease can be transmitted by blood transfusion. However, the exact kinetics of blood infectivity and the blood fraction carrying infectivity have not yet been identified. STUDY DESIGN AND METHODS: Simian PrP(c) epitopes were mapped by flow cytometry using monoclonal antibodies (MoAbs). A whole blood/no wash protocol was established, validated, and applied to investigate peripheral blood cell-associated PrP(c) expression profiles in bovine spongiform encephalopathy (BSE)-infected cynomolgus monkeys and age-/sex-matched controls. In addition, physiologic expression patterns on blood cells and in lymphoid tissues were determined. RESULTS: In BSE-infected macaques, blood lymphocyte-associated PrP(c) fluorescence gradually increased years before the onset of clinical signs (p(F test) < 0.0001). The increase in fluorescence intensity was detectable with MoAb 12F10, whereas we failed to detect an increase with 3F4. In parallel, plasma concentrations of soluble CD230 also increased. Centrifugation of lymphocytes almost completely eliminated differences between infected and noninfected animals, most likely caused by a partial loss in cell-associated CD230 into the plasma supernatant. CONCLUSION: Blood lymphocytes from asymptomatically infected as well as diseased macaques were characterized by increased CD230 fluorescence, and phosphatidylinositol-phospholipase C-resistant PrP molecules contributed at least partially to this increase. Conformational changes within PrP(c) molecules may be the underlying mechanism for the increased PrP(c) fluorescence. This cell-associated phenomenon contributed at least partially to an increase in soluble plasma-derived PrP(c) levels. It is not yet known whether these changes reflect infectivity.

Time-course studies of 14-3-3 protein isoforms in cerebrospinal fluid and brain of primates after oral or intracerebral infection with bovine spongiform encephalopathy agent.

Experimental transmission of bovine spongiform encephalopathy (BSE) to cynomolgus monkeys (Macaca fascicularis) is an animal model for variant Creutzfeldt-Jakob disease (vCJD). The presence of 14-3-3 proteins in cerebrospinal fluid (CSF) samples indicates neuronal destruction and is therefore used as a clinical biomarker. However, time-course studies using 14-3-3 proteins have not been performed until now in simian vCJD. The main goals of this study were to determine isoform patterns, to examine kinetics and to correlate the clinical course with the occurrence of this biomarker in simian vCJD. In monkeys dosed intracerebrally with BSE, the earliest clinical sign of illness was a drop in body weight that was detected months before the onset of mild neurological signs. Macaques dosed orally or intracerebrally with BSE developed neurological signs 4.3 (3.7-4.6) and 4.8 (2.9-6.0) years post-infection, respectively. 14-3-3beta- and -gamma-positive CSF samples were found around the time of onset of mild neurological signs, but not earlier. In contrast, 14-3-3epsilon and -eta isoforms were not detectable. 14-3-3 levels increased with time and were positively correlated with the degree of neurological symptoms. Post-mortem examination of brain samples revealed a positive correlation between PrP res and 14-3-3epsilon levels. Interestingly, florid plaques characteristic of human vCJD could not be detected in diseased monkeys. It was concluded that analysis of 14-3-3 proteins in CSF is a reliable tool to characterize the time course of brain degeneration in simian vCJD. However, there are differences in the clinical course between orally and intracerebrally infected animals that may influence the detection of other biomarkers.

Engineered CD4- and CXCR4-using simian immunodeficiency virus from African green monkeys is neutralization sensitive and replicates in nonstimulated lymphocytes.

During human immunodeficiency virus type 1 (HIV-1) infection, disease progression correlates with the occurrence of variants using the coreceptor CXCR4 for cell entry. In contrast, apathogenic simian immunodeficiency virus (SIV) from African green monkeys (SIVagm), specifically the molecular virus clone SIVagm3mc, uses CCR5, Bob, and Bonzo as coreceptors throughout the course of infection. The influence of an altered coreceptor usage on SIVagm3mc replication was studied in vitro and in vivo. The putative coreceptor binding domain, the V3 region of the surface envelope (SU) glycoprotein, was replaced by the V3 loop of a CD4- and CXCR4-tropic HIV-1 strain. The resulting virus, termed SIVagm3-X4mc, exclusively used CD4 and CXCR4 for cell entry. Consequently, its in vitro replication was inhibited by SDF-1, the natural ligand of CXCR4. Surprisingly, SIVagm3-X4mc was able to replicate in vitro not only in interleukin-2- and phytohemagglutinin-stimulated but also in nonstimulated peripheral blood mononuclear cells (PBMCs) from nonhuman primates. After experimental infection of two pig-tailed macaques with either SIVagm3-X4mc or SIVagm3mc, the coreceptor usage was maintained during in vivo replication. Cell-associated and plasma viral loads, as well as viral DNA copy numbers, were found to be comparable between SIVagm3mc and SIVagm 3-X4mc infections, and no pathological changes were observed up to 14 months postinfection. Interestingly, the V3 loop exchange rendered SIVagm3-X4mc susceptible to neutralizing antibodies present in the sera of SIVagm3-X4mc- and SIVagm3mc-infected pig-tailed macaques. Our study describes for the first time a successful exchange of a V3 loop in nonpathogenic SIVagm resulting in CD4 and CXCR4 usage and modulation of virus replication in nonstimulated PBMCs as well as sensitivity toward neutralization.

Immunological changes in simian immunodeficiency virus (SIV(agm))-infected African green monkeys (AGM): expanded cytotoxic T lymphocyte, natural killer and B cell subsets in the natural host of SIV(agm).

The African green monkey (AGM) model system for simian immunodeficiency virus (SIV(agm)) has been used to examine why prolonged infection with the relevant virus does not result in the development of immunodeficiency in its natural host. Blood lymphocyte subset values were determined in uninfected (n=88) and naturally SIV(agm)-infected AGMs (n=74). A number of blood cell subsets, such as CD8alpha(+)CD3(+)CD28(neg), CD8alpha(+)CD3(neg) and CD20(+) cells, were expanded significantly in clinically asymptomatic animals carrying a relatively high plasma load of viral RNA (10(4)-10(7) RNA copies/ml plasma). The expanded CD8alpha(+)CD3(+)CD28(neg) subpopulation (1094 +/- 986 cells/microl blood in infected animals versus 402 +/- 364 cells/microl blood, P=0.03) comprised cells that resembled terminally differentiated effector CD8 T cells (CD27(neg) and CD11a(+)). In SIV(agm)-infected animals, the expanded CD8alpha(+)CD3(neg) cell subset shared identity with the CD16(+) population (natural killer cells). These results demonstrate for the first time that apathogenic SIV(agm) infection causes significant changes in the immune system of its natural host. Although previous studies had indicated that noncytotoxic mechanisms might play an important role in the suppression of virus replication in the natural host of SIV(agm), this study sheds new light on the possible role of cytotoxic T lymphocytes, the innate immune system and double-positive T helper cells (CD4(+)CD8alpha(+)CD3(+)) in suppressing virus replication in this animal model of AIDS.