Susceptibility of Pallid Sturgeon to viral hemorrhagic septicemia virus genotype IVb.

OBJECTIVE: Viral hemorrhagic septicemia virus (VHSV) is an aquatic rhabdovirus causing severe disease in freshwater and saltwater fish species. The susceptibility of endangered Pallid Sturgeon Scaphirhynchus albus to VHSV genotype IVb (VHSV-IVb) infection was investigated. METHODS: An in vitro assessment using two Pallid Sturgeon cell lines derived from skin and spleen tissue and in vivo evaluation of juvenile Pallid Sturgeon after exposure to VHSV-IVb were performed. RESULT: Plaque assay and RT-PCR results confirmed VHSV-IVb replication in Pallid Sturgeon cell lines. Sturgeon were also susceptible to VHSV-IVb infection after immersion and injection exposures during laboratory experiments. However, after widespread mortality occurred in all treatment groups, including negative control fish, it was determined that the Pallid Sturgeon stock fish were infected with Missouri River sturgeon iridovirus (MRSIV) prior to experimental challenge. Nevertheless, mortalities were equal or higher among VHSV-exposed fish than among negative controls (MRSIV infected), and histopathological assessments indicated reduced hematopoietic cells in spleen and kidney tissues and hemorrhage in the gastrointestinal organs only in fish from the VHSV treatment. CONCLUSION: These results indicate that Pallid Sturgeon is a susceptible host for VHSV-IVb, but the degree of pathogenicity was confounded by the underlying MRSIV infection. Research comparing susceptibility of specific pathogen-free and MRSIV-infected fish to VHSV-IVb is needed to accurately assess the vulnerability of Pallid Sturgeon to VHSV-IVb.

B cell clonal lineage alterations upon recombinant HIV-1 envelope immunization of rhesus macaques.

Broadly neutralizing HIV-1 antibodies (bNAbs) isolated from infected subjects display protective potential in animal models. Their elicitation by immunization is thus highly desirable. The HIV-1 envelope glycoprotein (Env) is the sole viral target of bnAbs, but is also targeted by binding, non-neutralizing antibodies. Env-based immunogens tested so far in various animal species and humans have elicited binding and autologous neutralizing antibodies but not bNAbs (with a few notable exceptions). The underlying reasons for this are not well understood despite intensive efforts to characterize the binding specificities of the elicited antibodies; mostly by employing serologic methodologies and monoclonal antibody isolation and characterization. These approaches provide limited information on the ontogenies and clonal B cell lineages that expand following Env-immunization. Thus, our current understanding on how the expansion of particular B cell lineages by Env may be linked to the development of non-neutralizing antibodies is limited. Here, in addition to serological analysis, we employed high-throughput BCR sequence analysis from the periphery, lymph nodes and bone marrow, as well as B cell- and antibody-isolation and characterization methods, to compare in great detail the B cell and antibody responses elicited in non-human primates by two forms of the clade C HIV Env 426c: one representing the full length extracellular portion of Env while the other lacking the variable domains 1, 2 and 3 and three conserved N-linked glycosylation sites. The two forms were equally immunogenic, but only the latter elicited neutralizing antibodies by stimulating a more restricted expansion of B cells to a narrower set of IGH/IGK/IGL-V genes that represented a small fraction (0.003-0.02%) of total B cells. Our study provides new information on how Env antigenic differences drastically affect the expansion of particular B cell lineages and supports immunogen-design efforts aiming at stimulating the expansion of cells expressing particular B cell receptors.

Diverse recombinant HIV-1 Envs fail to activate B cells expressing the germline B cell receptors of the broadly neutralizing anti-HIV-1 antibodies PG9 and 447-52D.

UNLABELLED: Broadly neutralizing antibodies (bNAbs) against HIV-1 are generated during HIV-1-infection but have not yet been elicited by immunization with recombinant forms of the viral envelope glycoprotein (Env; the target of anti-HIV-1 neutralizing antibodies). A particular type of bNAb targets the CD4-binding site (CD4-BS) region of Env. These antibodies are derived from a limited number of VH/VL genes and can bind to and neutralize diverse HIV-1 strains. Recent reports have demonstrated the limited potential of Env to activate B cells expressing the germline B cell receptor (BCR) forms of anti-CD4-BS bNAbs. A potential reason for the lack of elicitation of anti-CD4-BS bNAbs by Env immunogens is the absence of stimulation of naive B cells expressing the germline BCRs of such antibodies. Several bNAbs have been isolated from HIV-1-infected subjects that target other structurally conserved regions of Env. How frequently Env immunogens stimulate the germline BCRs that give rise to bNAbs that target Env regions other than the CD4-BS is not well understood. Here, we investigated the interactions between diverse Envs and the BCRs of known bNAbs targeting not only the CD4-BS but also conserved elements of the second and third variable Env regions. Our results indicate that Env is generally ineffective in engaging germline BCRs of bNAbs irrespective of their epitope target. Potentially, this is the result of viral evolutionary mechanisms adopted to escape broadly neutralizing antibody responses. Our results also suggest that a single Env capable of activating germline BCRs that target distinct Env epitopes will be very difficult to identify or to design. IMPORTANCE: Broadly neutralizing antibodies against HIV-1 are thought to be an important component of the immune responses that a successful vaccine should elicit. Broadly neutralizing antibodies are generated by a subset of those infected by HIV-1, but so far, they have not been generated by immunization with recombinant Envelope (Env, the target of anti-HIV-1 neutralizing antibodies). Here, we provide evidence that the inability of Env to elicit the production of broadly neutralizing antibodies is due to the inability of diverse Envs to engage the germline B cell receptor forms of known broadly neutralizing antibodies.

Differences in Allelic Frequency and CDRH3 Region Limit the Engagement of HIV Env Immunogens by Putative VRC01 Neutralizing Antibody Precursors.

Elicitation of broadly neutralizing antibodies remains a long-standing goal of HIV vaccine research. Although such antibodies can arise during HIV-1 infection, gaps in our knowledge of their germline, pre-immune precursor forms, as well as on their interaction with viral Env, limit our ability to elicit them through vaccination. Studies of broadly neutralizing antibodies from the VRC01-class provide insight into progenitor B cell receptors (BCRs) that could develop into this class of antibodies. Here, we employed high-throughput heavy chain variable region (VH)/light chain variable region (VL) deep sequencing, combined with biophysical, structural, and modeling antibody analyses, to interrogate circulating potential VRC01-progenitor BCRs in healthy individuals. Our study reveals that not all humans are equally predisposed to generate VRC01-class antibodies, not all predicted progenitor VRC01-expressing B cells can bind to Env, and the CDRH3 region of germline VRC01 antibodies influence their ability to recognize HIV-1. These findings will be critical to the design of optimized immunogens that should consider CDRH3 interactions.

Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice.

VRC01-class broadly neutralizing HIV-1 antibodies protect animals from experimental infection and could contribute to an effective vaccine response. Their predicted germline forms (gl) bind Env inefficiently, which may explain why they are not elicited by HIV-1 Env-immunization. Here we show that an optimized Env immunogen can engage multiple glVRC01-class antibodies. Furthermore, this immunogen activates naive B cells expressing the human germline heavy chain of 3BNC60, paired with endogenous mouse light chains in vivo. To address whether it activates B cells expressing the fully humanized gl3BNC60 B-cell receptor (BCR), we immunized mice carrying both the heavy and light chains of gl3BNC60. B cells expressing this BCR display an autoreactive phenotype and fail to respond efficiently to soluble forms of the optimized immunogen, unless it is highly multimerized. Thus, specifically designed Env immunogens can activate naive B cells expressing human BCRs corresponding to precursors of broadly neutralizing HIV-1 antibodies even when the B cells display an autoreactive phenotype.

Soluble HIV-1 envelope immunogens derived from an elite neutralizer elicit cross-reactive V1V2 antibodies and low potency neutralizing antibodies.

We evaluated four gp140 Envelope protein vaccine immunogens that were derived from an elite neutralizer, subject VC10042, whose plasma was able to potently neutralize a wide array of genetically distinct HIV-1 isolates. We sought to determine whether soluble Envelope proteins derived from the viruses circulating in VC10042 could be used as immunogens to elicit similar neutralizing antibody responses by vaccination. Each gp140 was tested in its trimeric and monomeric forms, and we evaluated two gp140 trimer vaccine regimens in which adjuvant was supplied at all four immunizations or at only the first two immunizations. Interestingly, all four Envelope immunogens elicited high titers of cross-reactive antibodies that recognize the variable regions V1V2 and are potentially similar to antibodies linked with a reduced risk of HIV-1 acquisition in the RV144 vaccine trial. Two of the four immunogens elicited neutralizing antibody responses that neutralized a wide array of HIV-1 isolates from across genetic clades, but those responses were of very low potency. There were no significant differences in the responses elicited by trimers or monomers, nor was there a significant difference between the two adjuvant regimens. Our study identified two promising Envelope immunogens that elicited anti-V1V2 antibodies and broad, but low potency, neutralizing antibody responses.

Molecular identification of erythrocytic necrosis virus (ENV) from the blood of Pacific herring (Clupea pallasii).

Viral erythrocytic necrosis (VEN) is a condition affecting the red blood cells of more than 20 species of marine and anadromous fishes in the North Atlantic and North Pacific Oceans. Among populations of Pacific herring (Clupea pallasii) on the west coast of North America the disease causes anemia and elevated mortality in periodic epizootics. Presently, VEN is diagnosed by observation of typical cytoplasmic inclusion bodies in stained blood smears from infected fish. The causative agent, erythrocytic necrosis virus (ENV), is unculturable and a presumed iridovirus by electron microscopy. In vivo amplification of the virus in pathogen-free laboratory stocks of Pacific herring with subsequent virus concentration, purification, DNA extraction, and high-throughput sequencing were used to obtain genomic ENV sequences. Fragments with the highest sequence identity to the family Iridoviridae were used to design four sets of ENV-specific polymerase chain reaction (PCR) primers. Testing of blood and tissue samples from experimentally and wild infected Pacific herring as well as DNA extracted from other amphibian and piscine iridoviruses verified the assays were specific to ENV with a limit of detection of 0.0003 ng. Preliminary phylogenetic analyses of a 1448 bp fragment of the putative DNA polymerase gene supported inclusion of ENV in a proposed sixth genus of the family Iridoviridae that contains other erythrocytic viruses from ectothermic hosts. This study provides the first molecular evidence of ENV's inclusion within the Iridoviridae family and offers conventional PCR assays as a means of rapidly surveying the ENV-status of wild and propagated Pacific herring stocks.

HIV antibodies. Antigen modification regulates competition of broad and narrow neutralizing HIV antibodies.

Some HIV-infected individuals develop broadly neutralizing antibodies (bNAbs), whereas most develop antibodies that neutralize only a narrow range of viruses (nNAbs). bNAbs, but not nNAbs, protect animals from experimental infection and are likely a key component of an effective vaccine. nNAbs and bNAbs target the same regions of the viral envelope glycoprotein (Env), but for reasons that remain unclear only nNAbs are elicited by Env immunization. We show that in contrast to germline-reverted (gl) bNAbs, glnNAbs recognized diverse recombinant Envs. Moreover, owing to binding affinity differences, nNAb B cell progenitors had an advantage in becoming activated and internalizing Env compared with bNAb B cell progenitors. We then identified an Env modification strategy that minimized the activation of nNAb B cells targeting epitopes that overlap those of bNAbs.

Expression kinetics of key genes in the early innate immune response to Great Lakes viral hemorrhagic septicemia virus IVb infection in yellow perch (Perca flavescens).

The recently discovered strain of viral hemorrhagic septicemia virus, VHSV-IVb, represents an example of the introduction of an extremely pathogenic rhabdovirus capable of infecting a wide variety of new fish species in a new host-environment. The goal of the present study was to delineate the expression kinetics of key genes in the innate immune response relative to the very early stages of VHSV-IVb infection using the yellow perch (Perca flavescens) as a model. Administration of VHSV-IVb by IP-injection into juvenile yellow perch resulted in 84% cumulative mortality, indicating their high susceptibility to this disease. In fish sampled in the very early stages of infection, a significant up-regulation of Mx gene expression in the liver, as well as IL-1ß and SAA activation in the head kidney, spleen, and liver was directly correlated to viral load. The potential down-regulation of Mx in the hematopoietic tissues, head kidney and spleen, may represent a strategy utilized by the virus to increase replication.

Kinetics of viral load and erythrocytic inclusion body formation in Pacific herring artificially infected with erythrocytic necrosis virus.

Viral erythrocytic necrosis (VEN) is a condition that affects marine and anadromous fish species, including herrings and salmonids, in the Atlantic and Pacific oceans. Infection is frequently associated with severe anemia and causes episodic mortality among wild and hatchery fish when accompanied by additional stressors; VEN can be presumptively diagnosed by (1) light microscopic identification of a single characteristic-a round, magenta-colored, 0.8-µm-diameter inclusion body (IB) within the cytoplasm of erythrocytes and their precursors on Giemsa-stained blood films; or (2) observation (via transmission electron microscopy [TEM]) of the causative iridovirus, erythrocytic necrosis virus (ENV), within erythrocytes or their precursors. To better understand the kinetics of VEN, specific-pathogen-free Pacific herring Clupea pallasii were infected with ENV by intraperitoneal injection. At 1, 4, 7, 10, 14, 21, and 28 d postexposure, samples of blood, spleen, and kidney were collected and assessed (1) via light microscopy for the number of intracytoplasmic IBs in blood smears and (2) via TEM for the number of virions within erythrocytes. The mean prevalence of intracytoplasmic IBs in the blood cells increased from 0% at 0-4 d postexposure to 94% at 28 d postexposure. Viral load within circulating red blood cells peaked at 7 d postexposure, fell slightly, and then reached a plateau. However, blood cells observed within the kidney and spleen tissues demonstrated high levels of ENV between 14 and 28 d postexposure. The results indicate that the viral load within erythrocytes does not correlate well with IB prevalence and that the virus can persist in infected fish for more than 28 d.