Species-specific host factors rather than virus-intrinsic virulence determine primate lentiviral pathogenicity.

HIV-1 causes chronic inflammation and AIDS in humans, whereas related simian immunodeficiency viruses (SIVs) replicate efficiently in their natural hosts without causing disease. It is currently unknown to what extent virus-specific properties are responsible for these different clinical outcomes. Here, we incorporate two putative HIV-1 virulence determinants, i.e., a Vpu protein that antagonizes tetherin and blocks NF-κB activation and a Nef protein that fails to suppress T cell activation via downmodulation of CD3, into a non-pathogenic SIVagm strain and test their impact on viral replication and pathogenicity in African green monkeys. Despite sustained high-level viremia over more than 4 years, moderately increased immune activation and transcriptional signatures of inflammation, the HIV-1-like SIVagm does not cause immunodeficiency or any other disease. These data indicate that species-specific host factors rather than intrinsic viral virulence factors determine the pathogenicity of primate lentiviruses.

The ectodomain of rabies virus glycoprotein determines dendritic cell activation.

The immune evasion of wild-type (wt) rabies virus (RABV) has been attributed to its glycoprotein (G), particularly to their inefficiency to bind/enter into dendritic cells (DCs). However, the domain responsible for G-mediated DC activation is not clear. In the present study, attempts were made to map the domain(s) on the G involved in differential DC activation using laboratory-adapted and wt viruses. Recombinant RABVs with exchange in each of the structural domains such as signal peptide (sp), ectodomain (et), transmembrane domain (tm), cytoplasmic tail (ct) of the G between wt and laboratory-adapted strains were constructed. Characterizations of these recombinant RABVs show that the viruses containing the sp, tm and ct from the wt G are capable of growing in high titer by efficient cell-to-cell spread, similar to laboratory-adapted virus. On the other hand, recombinant virus containing the et domain from wt G was inefficient in cell-to-cell spread and grew in lower levels, similar to the wt RABV. Analysis of DC activation shows that viruses containing sp and tm from wt G are efficient in binding to and activating DCs. However, viruses containing the et domain from wt G are incompetent in binding to and activating DCs. Analysis of the G expression in the infected cells suggests that the level of G expression is regulated solely by the ct domain, indicating the level of G expression and DC activation are governed by different domains. Together, our results demonstrate that G-mediated DC activation is regulated by the et domain while the level of G expression by the ct domain.

Differential Host Immune Responses after Infection with Wild-Type or Lab-Attenuated Rabies Viruses in Dogs.

METHODOLOGY/PRINCIPAL FINDINGS: The experimental infection of dogs with TriGAS induced high levels of VNA in the serum, whereas wt RABV infection did not. Dogs infected with TriGAS developed antibodies against the virus including its glycoprotein, whereas dogs infected with DRV-NG11 only developed rabies antibodies that are presumably specific for the nucleoprotein, (N) and not the glycoprotein (G). We show that infection with TriGAS induces early activation of B cells in the draining lymph nodes and persistent activation of DCs and B cells in the blood. On the other hand, infection with DRV-NG11 fails to induce the activation of DCs and B cells and further reduces CD4 T cell production. Further, we show that intrathecal (IT) immunization of TriGAS not only induced high levels of VNA in the serum but also in the CSF while intramuscular (IM) immunization of TriGAS induced VNA only in the serum. In addition, high levels of total protein and WBC were detected in the CSF of IT immunized dogs, indicating the transient enhancement of blood-brain barrier (BBB) permeability, which is relevant to the passage of immune effectors from periphery into the CNS. CONCLUSIONS/SIGNIFICANCE: IM infection of dogs with TriGAS induced the production of serum VNA whereas, IT immunization of TriGAS in dogs induces high levels of VNA in the periphery as well as in the CSF and transiently enhances BBB permeability. In contrast, infection with wt DRV-NG11 resulted in the production of RABV-reactive antibodies but VNA and antibodies specific for G were absent. As a consequence, all of the dogs infected with wt DRV-NG11 succumbed to rabies. Thus the failure to activate protective immunity is one of the important features of RABV pathogenesis in dogs.

The inability of wild-type rabies virus to activate dendritic cells is dependent on the glycoprotein and correlates with its low level of the de novo-synthesized leader RNA.

UNLABELLED: Dendritic cells (DCs) are the most efficient antigen-presenting cells, playing a key role in the adaptive immune responses to viral infections. Our studies demonstrate that wild-type (wt) rabies virus (RABV) does not activate DCs. Adoptive transfer of DCs primed with wt RABV did not activate DCs, stimulate virus neutralizing antibodies (VNA), or protect recipients against challenge. However, adoptive transfer of DCs primed with laboratory-attenuated RABV resulted in DC activation, production of VNA, and protection against challenge. In vitro studies with recombinant RABV (laboratory-attenuated RABV expressing the glycoprotein or the phosphoprotein from wt RABV) demonstrate that DC activation is dependent on the glycoprotein and involves the IPS-1 pathway. Furthermore, binding to and entry into DCs by wt RABV is severely blocked, and the copy number of de novo-synthesized leader RNA was two logs lower in DCs infected with the wt than in DCs treated with laboratory-attenuated RABV. However, transient transfection of DCs with synthesized leader RNA from either wt or attenuated RABV is capable of activating DCs in a dose-dependent manner. Thus, the inability of wt RABV to activate DCs correlates with its low level of the de novo-synthesized leader RNA. IMPORTANCE: Rabies remains a public health threat, with more than 55,000 fatalities each year around the world. Since DCs play a key role in the adaptive immune responses to viral infections, we investigated the ability of rabies virus (RABV) to activate DCs. It was found that the adoptive transfer of DCs primed with wt RABV did not activate DCs, stimulate VNA, or protect mice against lethal challenge. However, laboratory-attenuated RABV mediates the activation of DCs via the IPS-1 pathway and is glycoprotein dependent. We further show that wt RABV evades DC-mediated immune activation by inefficient binding/entry into DCs and as a result of a reduced level of de novo-synthesized leader RNA. These findings may have important implications in the development of efficient rabies vaccines.

Presence of virus neutralizing antibodies in cerebral spinal fluid correlates with non-lethal rabies in dogs.

BACKGROUND: Rabies is traditionally considered a uniformly fatal disease after onset of clinical manifestations. However, increasing evidence indicates that non-lethal infection as well as recovery from flaccid paralysis and encephalitis occurs in laboratory animals as well as humans. METHODOLOGY/PRINCIPAL FINDINGS: Non-lethal rabies infection in dogs experimentally infected with wild type dog rabies virus (RABV, wt DRV-Mexico) correlates with the presence of high level of virus neutralizing antibodies (VNA) in the cerebral spinal fluid (CSF) and mild immune cell accumulation in the central nervous system (CNS). By contrast, dogs that succumbed to rabies showed only little or no VNA in the serum or in the CSF and severe inflammation in the CNS. Dogs vaccinated with a rabies vaccine showed no clinical signs of rabies and survived challenge with a lethal dose of wild-type DRV. VNA was detected in the serum, but not in the CSF of immunized dogs. Thus the presence of VNA is critical for inhibiting virus spread within the CNS and eventually clearing the virus from the CNS. CONCLUSIONS/SIGNIFICANCE: Non-lethal infection with wt RABV correlates with the presence of VNA in the CNS. Therefore production of VNA within the CNS or invasion of VNA from the periphery into the CNS via compromised blood-brain barrier is important for clearing the virus infection from CNS, thereby preventing an otherwise lethal rabies virus infection.

Recombinant rabies viruses expressing GM-CSF or flagellin are effective vaccines for both intramuscular and oral immunizations.

Our previous studies indicated that recombinant rabies viruses (rRABV) expressing chemokines or cytokines (including GM-CSF) could enhance the immunogenicity by recruiting and/or activating dendritic cells (DC). In this study, bacterial flagellin was cloned into the RABV genome and recombinant virus LBNSE-Flagellin was rescued. To compare the immunogenicity of LBNSE-Flagellin with recombinant virus expressing GMCSF (LBNSE-GMCSF), mice were immunized with each of these rRABVs by intramuscular (i.m.) or oral route. The parent virus (LBNSE) without expression of any foreign molecules was included for comparison. The i.m.-immunized mice were bled at three weeks after the immunization for the measurement of virus neutralizing antibody (VNA) and then challenged with 50 LD50 challenge virus standard (CVS-24). Orally immunized mice were boosted after three weeks and then bled and challenged one week after the booster immunization. It was found that both LBNSE-GMCSF and LBNSE-Flagellin recruited/activated more DCs and B cells in the periphery, stimulated higher levels of adaptive immune responses (VNA), and protected more mice against challenge infection than the parent virus LBNSE in both the i.m. and the orally immunized groups. Together, these studies suggest that recombinant RABV expressing GM-CSF or flagellin are more immunogenic than the parent virus in both i.m. and oral immunizations.

Complete genome sequence of a street rabies virus isolated from a dog in Nigeria.

A canine rabies virus (RABV) was isolated from a trade dog in Nigeria. Its entire genome was sequenced and found to be closely related to canine RABVs circulating in Africa. Sequence comparison indicates that the virus is closely related to the Africa 2 RABV lineage. The virus is now termed DRV-NG11.

A novel rabies vaccine based on a recombinant parainfluenza virus 5 expressing rabies virus glycoprotein.

Untreated rabies virus (RABV) infection leads to death. Vaccine and postexposure treatment have been effective in preventing RABV infection. However, due to cost, rabies vaccination and treatment have not been widely used in developing countries. There are 55,000 human death caused by rabies annually. An efficacious and cost-effective rabies vaccine is needed. Parainfluenza virus 5 (PIV5) is thought to contribute to kennel cough, and kennel cough vaccines containing live PIV5 have been used in dogs for many years. In this work, a PIV5-vectored rabies vaccine was tested in mice. A recombinant PIV5 encoding RABV glycoprotein (G) (rPIV5-RV-G) was administered to mice via intranasal (i.n.), intramuscular (i.m.), and oral inoculation. The vaccinated mice were challenged with a 50% lethal challenge dose (LD(50)) of RABV challenge virus standard 24 (CVS-24) intracerebrally. A single dose of 10(6) PFU of rPIV5-RV-G was sufficient for 100% protection when administered via the i.n. route. The mice vaccinated with a single dose of 10(8) PFU of rPIV5-RV-G via the i.m. route showed very robust protection (90% to 100%). Intriguingly, the mice vaccinated orally with a single dose of 10(8) PFU of rPIV5-RV-G showed a 50% survival rate, which is comparable to the 60% survival rate among mice inoculated with an attenuated rabies vaccine strain, recombinant LBNSE. This is first report of an orally effective rabies vaccine candidate in animals based on PIV5 as a vector. These results indicate that rPIV5-RV-G is an excellent candidate for a new generation of recombinant rabies vaccine for humans and animals and PIV5 is a potential vector for oral vaccines.

Down-modulation of CD8αß is a fundamental activity of primate lentiviral Nef proteins.

It is well established that the Nef proteins of human and simian immunodeficiency viruses (HIV and SIV) modulate major histocompatibility complex class I (MHC-I) cell surface expression to protect infected cells against lysis by cytotoxic T lymphocytes (CTLs). Recent data supported the observation that Nef also manipulates CTLs directly by down-modulating CD8αß (J. A. Leonard, T. Filzen, C. C. Carter, M. Schaefer, and K. L. Collins, J. Virol. 85:6867-6881, 2011), but it remained unknown whether this Nef activity is conserved between different lineages of HIV and SIV. In this study, we examined a total of 42 nef alleles from 16 different primate lentiviruses representing most major lineages of primate lentiviruses, as well as nonpandemic HIV-1 strains and the direct precursors of HIV-1 (SIVcpz and SIVgor). We found that the vast majority of these nef alleles strongly down-modulate CD8ß in human T cells. Primate lentiviral Nefs generally interacted specifically with the cytoplasmic tail of CD8ß, and down-modulation of this receptor was dependent on the conserved dileucine-based motif and two adjacent acidic residues (DD/E) in the C-terminal flexible loop of SIV Nef proteins. Both of these motifs are known to be important for the interaction of HIV-1 Nef with AP-2, and they were also shown to be critical for down-modulation of CD4 and CD28, but not MHC-I, by SIV Nefs. Our results show that down-modulation of CD4, CD8ß, and CD28 involves largely overlapping (but not identical) domains and is most likely dependent on conserved interactions of primate lentiviral Nefs with cellular adaptor proteins. Furthermore, our data demonstrate that Nef-mediated down-modulation of CD8αß is a fundamental property of primate lentiviruses and suggest that direct manipulation of CD8+ T cells plays a relevant role in viral immune evasion.