Asynchronous onset of clinical disease in BSE-infected macaques.

To estimate the effect of the variability of prion disease onset on primary bovine spongiform encephalopathy transmission to humans, we studied 6 cynomolgus macaques. The preclinical incubation period was significantly prolonged in 2 animals, implying that onset of variant Creutzfeldt-Jacob disease in humans could be more diverse than previously expected.

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.

N-terminal myristoylation-dependent masking of neutralizing epitopes in the preS1 attachment site of hepatitis B virus.

BACKGROUNDS & AIMS: The N-terminally myristoylated preS1 domain of the large hepatitis B surface protein (LHBs) mediates specific attachment of hepatitis B virus (HBV) to hepatocytes. Its B-cell epitopes leading to neutralization of infectivity are not yet characterized. METHODS: We inserted C- and N-terminal preS1 peptides into the most immunogenic region of HBV core particles, therewith immunized Balb/c mice and determined binding properties and neutralization potential of resulting antibodies in vitro. RESULTS: The particles with preS1 inserts were highly immunogenic and the corresponding anti-preS antibodies strongly bound to HBV particles from chronic carriers infected with different HBV genotypes A-F. However, antibodies binding to the C-terminal part of preS1 did not neutralize HBV infectivity for susceptible hepatocyte cultures. In contrast, antibodies elicited by the complete N-terminal attachment site of preS1(2-48) strongly neutralized with an IC50<3µg/ml of total immunoglobulin. Interestingly, antibodies against the very N-terminal part of preS1(1-21) could not neutralize infectivity although this sequence contains the most conserved and essential part of the attachment site. These antibodies reacted well with non-myristoylated preS1 peptides but only weakly with myristoylated preS1 peptides, natural HBsAg or HBV. CONCLUSIONS: N-terminal myristic acid obviously favors a topology of LHBs that makes the most essential part of the preS1 attachment site inaccessible for neutralizing antibodies, whereas antibodies to neighbouring sequences neutralized very well. Thus, addition of the preS1(2-48) peptide in a highly immunogenic form to the current hepatitis B vaccine may improve protective immunity and reduce selection of escape mutations.

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.

Better protective effects in rhesus macaques by combining systemic and mucosal application of a dual component vector vaccine after rectal SHIV89.6P challenge compared to systemic vaccination alone.

In this study we investigated the efficacy of a multigenic DNA prime/modified vaccinia Ankara (MVA)boost vaccine approach, followed by mucosal challenge with highly pathogenic simian-human immunodeficiency virus (SHIV) 89.6P, using different routes for vaccine delivery. After three times of DNA priming (SIVmac239, GagPol, and SHIV 89.6P Env) one vaccine group of monkeys was immunized with MVA systemically via intramuscular (IM) and intradermal (ID) application, and in another vaccine group the MVA booster immunization comprised the IM, ID, and atraumatic oral route. Although all vaccinees became infected after intra-rectal challenge with SHIV 89.6P, substantial protection as indicated by lower peak and set point viral loads and unambiguous preservation of CD4 T cells could be achieved. As we could only transiently detect low levels of neutralizing antibodies in some vaccinees, these antibodies did not seem to add to the protection in the vaccinees. Our results indicate that both preventive multigenic DNA prime/MVA booster immunization strategies promote the control of virus replication and protect from disease progression. We also demonstrated that combining mucosal and systemic vaccination mediated better protective effects compared to systemic vaccination alone.

Sustained conservation of CD4+ T cells in multiprotein triple modality-immunized rhesus macaques after intrarectal challenge with simian immunodeficiency virus.

As part of a European multicenter study designed to determine the optimal combination and order of a mixed-modality vaccine against acquired immunodeficiency syndrome, rhesus monkeys received a combination of three different vectors, all expressing the same Simian Immunodeficiency Virus (SIV) genes followed by mucosal challenge with highly pathogenic SIV. In the study reported here, animals were primed with DNA followed by one booster immunization with Semliki Forest Virus (SFV) and two immunizations with modified Vaccinia Ankara (MVA). To address the relevance of mucosal immunization, we compared systemic versus a combination of systemic and mucosal antigen application. Although all vaccinees became infected after intrarectal challenge with SIV, most (six of eight) were protected from profound loss of CD4+ cells. In addition, vaccinees showed lower viral loads than did controls (p < 0.05). Overall, these protective effects were more pronounced in those animals whose schedule included immunization via the mucosa. In summary, the vaccine regimen used here achieved one important criterion of efficacy: the suppression of disease development as indicated by conservation of CD4+ cells.

Separation of native prion protein (PrP) glycoforms by copper-binding using immobilized metal affinity chromatography (IMAC).

The conformational conversion of the normal cellular prion protein (PrPC) into the pathology-associated PrPSc isoform is a key event in TSEs (transmissible spongiform encephalopathies). The host PrPC molecule contains two N-linked glycosylation sites and binds copper under physiological conditions. In contrast with PrPC, PrPSc is insoluble in non-ionic detergents and does not bind to Cu2+ ions. Hence, we utilized copper binding to separate and characterize both PrP isoforms. Infected and uninfected murine brain and bovine stem brain specimens were treated with the mild non-ionic detergent n-octyl-beta-D-glucopyranoside (octylglucoside) to maintain the native PrP conformations during isolation. The solubilized homogenates were loaded on to Cu2+-saturated IMAC (immobilized metal affinity chromatography) columns and eluted using the chelating agent EDTA. Fractions were separated by SDS/PAGE and analysed by immunoblotting using anti-PrP monoclonal antibodies for glycosylation profiling. Whereas native PrPC and denatured PrPSc were retained by a Cu2+-loaded resin, native PrPSc and PrPres [PK (proteinase K)-resistant PrP] passed through the column. We demonstrate here that the IMAC technique is appropriate to isolate and partially purify PrPC from healthy brains in its native-like and biologically relevant glycosylated copper-binding forms. The IMAC technique is also well suited for the separation of native PrPC from aggregated PrPSc in infected brains. Our results indicate that in contrast with PrPSc in uninfected as well as infected brains, PrPC is predominantly present in the glycosylated forms.

Multiple antibody targets on herpes B glycoproteins B and D identified by screening sera of infected rhesus macaques with peptide microarrays.

Herpes B virus (or Herpesvirus simiae or Macacine herpesvirus 1) is endemic in many populations of macaques, both in the wild and in captivity. The virus elicits only mild clinical symptoms (if any) in monkeys, but can be transmitted by various routes, most commonly via bites, to humans where it causes viral encephalitis with a high mortality rate. Hence, herpes B constitutes a considerable occupational hazard for animal caretakers, veterinarians and laboratory personnel. Efforts are therefore being made to reduce the risk of zoonotic infection and to improve prognosis after accidental exposure. Among the measures envisaged are serological surveillance of monkey colonies and specific diagnosis of herpes B zoonosis against a background of antibodies recognizing the closely related human herpes simplex virus (HSV). 422 pentadecapeptides covering, in an overlapping fashion, the entire amino acid sequences of herpes B proteins gB and gD were synthesized and immobilized on glass slides. Antibodies present in monkey sera that bind to subsets of the peptide collection were detected by microserological techniques. With 42 different rhesus macaque sera, 114 individual responses to 18 different antibody target regions (ATRs) were recorded, 17 of which had not been described earlier. This finding may pave the way for a peptide-based, herpes B specific serological diagnostic test.

Upregulation of miRNA hsa-miR-342-3p in experimental and idiopathic prion disease.

The aim of our study was to analyze the differential expression of miRNAs in the brains of BSE-infected cynomolgus macaques as a model for Creutzfeldt-Jakob disease (CJD). MicroRNAs (miRNAs) are small noncoding RNAs regulating gene expression by mRNA targeting. Among other functions they contribute to neuronal development and survival. Recently, the lack of miRNA processing has been shown to promote neurodegeneration and deregulation of several miRNAs has been reported to be associated with Scrapie in mice. Therefore, we hypothesized that miRNAs are also regulated in response to human prion disease. We have applied miRNA-microarrays to identify deregulated miRNA candidates in brains of BSE-infected macaques. Shock-frozen brain sections of six BSE-infected and five non-infected macaques were used to validate regulated miRNA candidates by two independent qRT-PCR-based methods. Our study revealed significant upregulation of hsa-miR-342-3p and hsa-miR-494 in the brains of BSE-infected macaques compared to non-infected animals. In a pilot study we could show that hsa-miR-342-3p was also upregulated in brain samples of human type 1 and type 2 sporadic CJD. With respect to the reported regulation of this miRNA in Scrapie-infected mice, we propose that upregulation of hsa-miR-342-3p may be a general phenomenon in late stage prion disease and might be used as a novel marker for animal and human TSEs.

Susceptibility to Simian immunodeficiency virus ex vivo predicts outcome of a prime-boost vaccine after SIVmac239 challenge.

BACKGROUND: Efficacy assessment of AIDS vaccines relies both on preclinically challenging immunized monkeys with simian immunodeficiency virus (SIV) or monitoring infection rates in large human trials. Although conventional parameters of vaccine-induced immune responses do not completely predict outcome, existing methods for testing cellular immunity are sophisticated and difficult to establish in resource-limited settings. METHODS: We have used virus replication kinetics (VVR) on ConA-stimulated peripheral blood mononuclear cells from rhesus monkeys immunized with DNA replication-defective adenovirus vector expressing various SIV genes, as an ex vivo model, to mimic the effects of different immune effector functions on viral infection. RESULTS: VVR was attenuated by the immunization and correlated 2 weeks after first boost, with the number of interferon gamma-secreting cells and T-cell noncytotoxic antiviral responses. Importantly, VVR on the day of challenge but not interferon gamma responses correlated with viremia and with memory CD4+ T-cell measurements after SIVmac239 challenge. Similarly, T-cell noncytotoxic antiviral responses on the day of challenge correlated directly with memory CD4 T cell and inversely with plasma viremia after challenge. CONCLUSIONS: VVR thus served as a better predictor of protective capacity of the vaccine regimen in these monkeys. We suggest that VVR be considered in the evaluation of candidate AIDS vaccines in humans.

Mucosal prior to systemic application of recombinant adenovirus boosting is more immunogenic than systemic application twice but confers similar protection against SIV-challenge in DNA vaccine-primed macaques.

We investigated the immunogenicity and efficacy of a bimodal prime/boost vaccine regimen given by various routes in the Simian immunodeficiency virus (SIV) rhesus monkey model for AIDS. Twelve animals were immunized with SIV DNA-vectors followed by the application of a recombinant adenovirus (rAd5) expressing the same genes either intramuscularly (i.m.) or by oropharyngeal spray. The second rAd5-application was given i.m. All vaccinees plus six controls were challenged orally with SIVmac239 12 weeks post-final immunization. Both immunization strategies induced strong SIV Gag-specific IFN-gamma and T-cell proliferation responses and mediated a conservation of CD4(+) memory T-cells and a reduction of viral load during peak viremia following infection. Interestingly, the mucosal group was superior to the systemic group regarding breadth and strength of SIV-specific T-cell responses and exhibited lower vector specific immune responses. Therefore, our data warrant the inclusion of mucosal vector application in a vaccination regimen which makes it less invasive and easier to apply.

Prolonged survival of vaccinated macaques after oral SIVmac239 challenge regardless of viremia control in the chronic phase.

To evaluate the efficacy of a multigenic vaccine and its protective immunity in the SIVmac239 challenge model, 12 rhesus macaques were divided into two groups. The vaccine group was intramuscularly immunized with multigenic DNA and recombinant adenovirus vaccine, while the control group received buffers. At 16 weeks after the last immunization, all macaques were challenged orally with pathogenic SIVmac239. The mean plasma SIV RNA loads of the vaccine group were significantly lower than those of the placebo control group up to 16 weeks post-challenge. The vaccine-induced Gag-specific IFN-gamma ELISPOT T cell responses inversely correlated with the viral loads before the chronic phase. Two out of six vaccinated macaques with strong and sustained Gag-specific T cell responses showed viremia control and maintained CD4+ T cell percentage. However, the other four vaccinated macaques showed high viral loads and reduced level of CD4+ T cell percentages during the chronic phase, comparable to those in control macaques. Five out of six vaccinated macaques survived for more than 72 weeks, while five out of six controls died of an AIDS-related disease. Therefore, the vaccination conferred not only reduction of viral loads in a portion of vaccinated macaques (2/6), but also prolonged survival of all vaccinated macaques regardless of viremia control. Our results further suggest that new experimental approaches may be needed to assess protective effects from AIDS-associated disease in the immunized macaques after oral SIV challenge.

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.

Immunogenicity of a DNA prime and recombinant adenovirus boost regime significantly varies between rhesus macaques of Chinese and Indian origins.

BACKGROUND: Due to an ever increasing shortage of rhesus macaques of Indian origin (InR) that have been generally used for preclinical AIDS vaccine trials in non-human primates, demand is rising for Chinese rhesus macaques (ChR). However, the immunogenicity of an AIDS vaccine candidate has not been compared in parallel in both rhesus macaque subspecies. METHODS: ChR and InR were immunized with SIV/HIV DNA and adenovirus vaccine and their immune responses to SIV and HIV evaluated. RESULTS: SIV Gag- and Env-specific T-cell responses and SIV-specific lymphoproliferative responses measured in ChR were significantly weaker than those in InR (P < 0.05). By contrast, antibody responses to SIV Env, Tat, and Nef in ChR were stronger than those in InR (P < 0.05). CONCLUSIONS: Immunogenicity of an AIDS vaccine can vary significantly depending on the geographic origin implying genetic differences of macaques. This must be considered when describing and interpreting results of such vaccine studies.

Identification of prion protein binding proteins by combined use of far-Western immunoblotting, two dimensional gel electrophoresis and mass spectrometry.

The cellular prion protein (PrP(C)), a highly conserved glycoprotein predominantly expressed by neuronal cells, can convert into an abnormal isoform (PrP(Sc)) and provoke a transmissible spongiform encephalopathy. In spite of many studies, the physiological function of PrP(C) remains unknown. Recent findings suggest that PrP(C) is a multifunctional protein participating in several cellular processes. Using recombinant human PrP as a probe, we performed far-Western immunoblotting (protein overlay assay) to detect cellular PrP(C) interactors. Brain extracts of wild-type and PrP knockout mice were screened by far-Western immunoblotting for PrP-specific interactions. Subsequently, putative ligands were isolated by 2-DE and identified by MALDI-TOF MS, enabling identification of heterogeneous nuclear ribonucleoprotein A2/B1 and aldolase C as novel interaction partners of PrP(C). These data provide the first evidence of a molecule indicating a mechanism for the predicted involvement of PrP(C) in nucleic acid metabolisms. In summary, we have shown the successful combination of 2-DE with far-Western immunoblotting and MALDI-TOF MS for identification of new cellular binding partners of a known protein. Especially the application of this technique to investigate other neurodegenerative diseases is promising.

Expression profiles of endogenous retroviruses in Old World monkeys.

Human endogenous retroviruses (HERVs) are a major component of the human genome and an active part of the transcriptome. Some HERVs play vital biological roles, while others potentially contribute to diseases. Many HERVs are relatively new in the primate genome, having entered or expanded after the lineages leading to the platyrrhines (New World monkeys) and catarrhines (Old World monkeys and apes) separated. Most HERVs are active in at least some tissues, though tissue specificity is common for most elements. We analyzed multiple tissues from several Old World monkeys using retroviral pol-based DNA microarrays and quantitative PCR methods to determine their ERV expression profiles. The results demonstrate that while many ERVs are active in nonhuman primates, overall the tissue expression specificity is unique to each species. Most striking is that while the majority of HERVs analyzed in this study are expressed in human brain, almost none are expressed in Old World monkey brains or are only weakly expressed.

Reduction of viral loads by multigenic DNA priming and adenovirus boosting in the SIVmac-macaque model.

In this study, we investigated the ability of a multigenic SIV DNA prime/replication-defective adenovirus serotype 5 (rAd/SIV) boost regimen to induce SIV-specific immune responses and protection against intrarectal challenge with SIVmac251 in rhesus macaques. Four rhesus macaques were immunized intramuscularly three times at 8-week intervals with SIV DNA vaccine and boosted once with rAd/SIV vaccine Four control macaques received the same amount of mock plasmid DNA and mock adenovirus vector. While the SIV DNA vaccine included plasmids expressing a mutated human IL-12 gene (IL-12N222L) as well as SIVmac239 structural and regulatory genes, the rAd/SIV vaccine contained rAd vectors expressing SIVmac239 genes only. Immunization with SIV DNA vaccine alone induced SIV-specific IFN-gamma ELISPOT responses in only two of four vaccinated macaques, whereas all animals developed SIV-specific T-cell responses and Env- and Tat-specific antibody responses following the rAd/SIV vaccine boost. Upon intrarectal challenge with pathogenic SIVmac251, strong anamnestic Env-specific binding and neutralizing antibody responses were detected in the vaccinated macaques. Overall, the immunized macaques had lower peak and set-point viral loads than control macaques, suggesting that the induced immune responses play a role in the control of viremia. In addition, the loss of CD4+ T cells was delayed in the vaccinated macaques after challenge. These results indicate that the multigenic DNA prime-adenovirus boost immunization may be a promising approach in developing an effective AIDS vaccine.

Genome-wide targeted search for human specific and polymorphic L1 integrations.

Retroelements (REs) occupy up to 40% of the human genome. Newly integrated REs can change the pattern of expression of pre-existing host genes and therefore might play a significant role in evolution. In particular, human- and primate-specific REs could affect the divergence of the Hominoidea superfamily. A comparative genome-wide analysis of RE sites of integration, neighboring genes, and their regulatory interplay in human and ape genomes would be of help in understanding the impact of REs on evolution and genome regulation. We have developed a technique for the genome-wide comparison of the integrations of transposable elements in genomic DNAs of closely related species. The technique called targeted genome differences analysis (TGDA) is also useful for the detection of deletion/insertion polymorphisms of REs. The technique is based on an enhanced version of subtractive hybridization and does not require preliminary knowledge of the genome sequences under comparison. In this report, we describe its application to the detection and analysis of human specific L1 integrations and their polymorphisms. We obtained a library highly enriched in human-specific L1 insertions and identified 24 such new insertions. Many of these insertions are polymorphic in human populations. The total number of human-specific L1 inserts was estimated to be approximately 4000. The results suggest that TGDA is a universal method that can be successfully used for the detection of evolutionary and polymorphic markers in any closely related genomes.

Human-specific subfamilies of HERV-K (HML-2) long terminal repeats: three master genes were active simultaneously during branching of hominoid lineages.

Using 40 known human-specific LTR sequences, we have derived a consensus sequence for an evolutionary young HERV-K (HML-2) LTR family, which was named the HS family. In the human genome the HS family is represented by approximately 150-160 LTR sequences, 90% of them being human-specific (hs). The family can be subdivided into two subfamilies differing in five linked nucleotide substitutions: HS-a and HS-b of 5.8 and 10.3 Myr evolutionary ages, respectively. The HS-b subfamily members were transpositionally active both before the divergence of the human and chimpanzee ancestor lineages and after it in both lineages. The HS-a subfamily comprises only hs LTRs. These and other data strongly suggest that at least three "master genes" of HERV-K (HML-2) LTRs were active in the human ancestor lineage after the human-chimpanzee divergence. We also found hs HERV-K (HML-2) LTRs integrations in introns of 12 human genes and identified 13 new hs HERV-K (HML-2) LTRs.

A technique for genome-wide identification of differences in the interspersed repeats integrations between closely related genomes and its application to detection of human-specific integrations of HERV-K LTRs.

We have developed a method of targeted genomic difference analysis (TGDA) for genomewide detection of interspersed repeat integration site differences between closely related genomes. The method includes a whole-genome amplification of the flanks adjacent to target interspersed repetitive elements in both genomic DNAs under comparison, and subtractive hybridization (SH) of the selected amplicons. The potential of TGDA was demonstrated by the detection of differences in the integration sites of human endogenous retroviruses K (HERV-K) and related solitary long terminal repeats (LTRs) between the human and chimpanzee genomes. Of 55 randomly sequenced clones from a library enriched with human-specific integration (HSI) sites, 33 (60%) represented HSIs. All the human-specific (Hs) LTRs belong to two related evolutionarily young groups, suggesting simultaneous activity of two master genes in the hominid lineage. No deletion/insertion polymorphism was detected for the LTR HSIs for 25 unrelated caucasoid individuals. We also discuss the possible research applications for TGDA research.