Pathogenic Correlates of Simian Immunodeficiency Virus-Associated B Cell Dysfunction.

We compared and contrasted pathogenic (in pig-tailed macaques [PTMs]) and nonpathogenic (in African green monkeys [AGMs]) SIVsab infections to assess the significance of the B cell dysfunction observed in simian (SIV) and human immunodeficiency virus (HIV) infections. We report that the loss of B cells is specifically associated with the pathogenic SIV infection, while in the natural hosts, in which SIV is nonpathogenic, B cells rapidly increase in both lymph nodes (LNs) and intestine. SIV-associated B cell dysfunction associated with the pathogenic SIV infection is characterized by loss of naive B cells, loss of resting memory B cells due to their redistribution to the gut, increases of the activated B cells and circulating tissue-like memory B cells, and expansion of the B regulatory cells (Bregs). While circulating B cells are virtually restored to preinfection levels during the chronic pathogenic SIV infection, restoration is mainly due to an expansion of the "exhausted," virus-specific B cells, i.e., activated memory cells and tissue-like memory B cells. Despite of the B cell dysfunction, SIV-specific antibody (Ab) production was higher in the PTMs than in AGMs, with the caveat that rapid disease progression in PTMs was strongly associated with lack of anti-SIV Ab. Neutralization titers and the avidity and maturation of immune responses did not differ between pathogenic and nonpathogenic infections, with the exception of the conformational epitope recognition, which evolved from low to high conformations in the natural host. The patterns of humoral immune responses in the natural host are therefore more similar to those observed in HIV-infected subjects, suggesting that natural hosts may be more appropriate for modeling the immunization strategies aimed at preventing HIV disease progression. The numerous differences between the pathogenic and nonpathogenic infections with regard to dynamics of the memory B cell subsets point to their role in the pathogenesis of HIV/SIV infections and suggest that monitoring B cells may be a reliable approach for assessing disease progression.IMPORTANCE We report here that the HIV/SIV-associated B cell dysfunction (defined by loss of total and memory B cells, increased B regulatory cell [Breg] counts, and B cell activation and apoptosis) is specifically associated with pathogenic SIV infection and absent during the course of nonpathogenic SIV infection in natural nonhuman primate hosts. Alterations of the B cell population are not correlated with production of neutralizing antibodies, the levels of which are similar in the two species. Rapid progressive infections are associated with a severe impairment in SIV-specific antibody production. While we did not find major differences in avidity and maturation between the pathogenic and nonpathogenic SIV infections, we identified a major difference in conformational epitope recognition, with the nonpathogenic infection being characterized by an evolution from low to high conformations. B cell dysfunction should be considered in designing immunization strategies aimed at preventing HIV disease progression.

Emerging EPO and EPO receptor regulators and signal transducers.

As essential mediators of red cell production, erythropoietin (EPO) and its cell surface receptor (EPO receptor [EPOR]) have been intensely studied. Early investigations defined basic mechanisms for hypoxia-inducible factor induction of EPO expression, and within erythroid progenitors EPOR engagement of canonical Janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5), rat sarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (RAS/MEK/ERK), and phosphatidylinositol 3-kinase (PI3K) pathways. Contemporary genetic, bioinformatic, and proteomic approaches continue to uncover new clinically relevant modulators of EPO and EPOR expression, and EPO's biological effects. This Spotlight review highlights such factors and their emerging roles during erythropoiesis and anemia.

Evidence of early B-cell dysregulation in simian immunodeficiency virus infection: rapid depletion of naïve and memory B-cell subsets with delayed reconstitution of the naïve B-cell population.

Despite eliciting a robust antibody response in humans, several studies in human immunodeficiency virus (HIV)-infected patients have demonstrated the presence of B-cell deficiencies during the chronic stage of infection. While several explanations for the HIV-induced B-cell deficit have been proposed, a clear mechanistic understanding of this loss of B-cell functionality is not known. This study utilizes simian immunodeficiency virus (SIV) infection of rhesus macaques to assess B-cell population dynamics beginning at the acute phase and continuing through the chronic phase of infection. Flow cytometric assessment demonstrated a significant early depletion of both naïve and memory B-cell subsets in the peripheral blood, with differential kinetics for recovery of these populations. Furthermore, the altered numbers of naïve and memory B-cell subsets in these animals corresponded with increased B-cell activation and altered proliferation profiles during the acute phase of infection. Finally, all animals produced high titers of antibody, demonstrating that the measurement of virus-specific antibody responses was not an accurate reflection of alterations in the B-cell compartment. These data indicate that dynamic B-cell population changes in SIV-infected macaques arise very early after infection at the precise time when an effective adaptive immune response is needed.

Nonpathogenic simian immunodeficiency virus infection of sooty mangabeys is not associated with high levels of autologous neutralizing antibodies.

Simian immunodeficiency virus (SIV) infection of natural-host species, such as sooty mangabeys (SMs), is characterized by a high level of viral replication and a low level of generalized immune activation, despite evidence of an adaptive immune response. Here the ability of SIV-infected SMs to mount neutralizing antibodies (Nab) against autologous virus was compared to that of human immunodeficiency virus type 1 (HIV-1) subtype C-infected subjects. While high levels of Nab were observed in HIV-1 infection, samples obtained at comparable time points from SM exhibited relatively low titers of autologous Nab. Nevertheless, SM plasma with higher Nab titers also contained elevated peripheral CD4(+) T-cell levels, suggesting a potential immunologic benefit for SMs. These data indicate that AIDS resistance in these primates is not due to high Nab titers and raise the possibility that low levels of Nab might be an inherent feature of natural-host SIV infections.

Chemoselective synthesis and biological evaluation of arylated 2-(Trifluoromethyl) quinolines as nucleotide pyrophosphatase (NPPs) inhibitors.

A new approach to arylated 2-trifluoromethylquinolines based on novel regioselective Suzuki-Miyaura coupling reactions has been developed. Moreover, site-selective, chemo-selective amination reactions were performed. The new 2-trifluoromethylquinoline derivatives were tested as potential NPPs inhibitors and evaluated for their potential to inhibit two families of ecto-nucleotidases, i.e. NPPs and nucleoside triphosphate diphosphohydrolases (NTPDases). Several derivatives were active on a nanomolecular concentration. The results were validated based on docking studies to study the active binding site of the molecules.

Intermittent heat exposure and thirst in rats.

Adequate water intake, supporting both cardiovascular function and evaporative cooling, is a critical factor in mitigating the effects of heat waves, which are expected to increase with global warming. However, the regulation of water intake during periods of intermittent heat exposure is not well understood. In this study, the effects of access to water or no access during intermittent heat exposure were assessed using male Sprague-Dawley rats exposed to 37.5°C for 4 h/day. After 7 days of intermittent heat exposure, reductions in evaporative water loss were observed in all animals and reductions in water intake following heat exposure occurred as the days of heat exposure increased. Rats that were not allowed water during the 7 days of exposure had decreased rehydration levels, however, rats allowed access to water increased water intake during exposure and exhibited higher overall rehydration levels over the same time period. Peripheral administration of angiotensinII, mimicking activation of volemic thirst, or hypertonic saline solution, activating intracellular thirst, did not result in alteration of water intake in rats exposed to heat with access to water compared to control rats. In contrast, rats exposed to heat without access to water had reduced water intake after administration of hypertonic saline and increased water intake after administration of angiotensinIIcompared to control rats. These experiments demonstrate that thirst responses to intermittent heat exposure are altered by providing water during heat exposure and that intermittent heat exposure without access to water alters drinking responses to both intracellular and extracellular thirst challenges.

Human single-chain antibodies inhibit replication of human immunodeficiency virus type 1 (HIV-1).

The F240 human monoclonal antibody specifically recognizes the disulfide loop-bonded immunodominant epitope of gp41 spanning residues 592-606 and expressed broadly on HIV-1 primary isolates. Despite broad reactivity with native virions and HIV-infected cells, the antibody fails to neutralize infection. However, cytoplasmic expression of single-chain antibody (scFv) directed against gp41 of HIV-1 provides a rationale means to inhibit the maturation of envelope protein. The variable regions of the heavy chain and light chain of human monoclonal antibody were amplified by PCR and linked by a 15 amino acid (GGSGS)3 linker in an orientation of VL-linker-VH and retroviral expression vectors were constructed to simultaneously express F240 scFv and eGFP to facilitate selection of scFv-producing cells. Incorporation of a human immunoglobulin signal sequence directed secretion of the F240 scFv (s-scFv) while an otherwise identical vector lacked this sequence (scFv) resulting in intracellular expression of scFv. Transduced human CD4+ H9 T cells were challenged with HIV. While both secreted and nonsecreted F240 scFv inhibited viral production, secretory F240 scFv was more potent. Thus, this novel approach to direct expression of a nonneutralizing scFv using the Ig signal sequence suggests that targeted therapy using antibodies to conserved, highly expressed epitopes may result in a decrease in viral production due to a reduction of viral assembly and/or transport and expression.

Expression and functional activity of isotype and subclass switched human monoclonal antibody reactive with the base of the V3 loop of HIV-1 gp120.

Immunoglobulins undergo isotype switching in response to antigenic stimulation. The C(H) domains, in particular the hinge region, impose structural constraints on the interaction of antibody with antigen, especially multivalent antigens such as HIV. We previously showed that switching the IgG1 anti-HIV human monoclonal antibody (HMAb) F105 to an IgG3 resulted in significantly enhanced neutralization of HIV. To further investigate the influence of isotype, including the functional activity of HMAbs switched to IgA, which may be important in mucosal defenses, isotype switched antibodies have been generated for the anti-V3 loop base IgG2 HMAb F425B4e8. Reactivity of the IgG1 antibody was greater than the parental IgG2 antibody for SF2 infected cells but less for primary isolate virions. In contrast, there was less reactivity of the IgG3 with either infected cells or virions. IgA reacted significantly more with infected cells and virions as compared to the IgG subclasses. In contrast to previous studies whereby IgG3 enhanced neutralization, comparable neutralization of primary isolate virus was observed for IgG subclasses (IgG1, IgG2, IgG3) and IgA. This may reflect differences in the exposure of epitopes recognized by the HMAb with antibody flexibility being important to neutralization by antibodies reactive with obscured epitopes (e.g., CD4 binding site). Further analysis of the in vitro activity of isotype or subclass switched antibodies, IgA in particular, alone and in combination with other HMAbs, will provide important information on the role of IgG subclass and IgA antibodies on protective immunity to HIV.

Binding and neutralization activity of human IgG1 and IgG3 from serum of HIV-infected individuals.

The IgG1 and IgG3 subclasses represent the predominant antibody response to viral infections, including HIV. IgG subclasses differ in their interaction with antigen and functional effects due to specific physiochemical features. With an elongated hinge, IgG3 antibodies tend to have more segmental flexibility, which can render the antibody more effective at interacting with antigen. We have previously shown that the change of the human anti-CD4-binding site monoclonal antibody F105 from IgG1 to IgG3 results in neutralization of a T cell line-adapted isolate (TCLA) resistant to neutralization by the parental IgG1. In the studies presented here, we have purified IgG1 and IgG3 subclasses from the sera of HIV-infected individuals and tested for immunoreactivity with and neutralization of HIV. Purified total IgG3 tended to have less relative reactivity and mediated relatively poorer neutralization of either laboratory or primary isolates. IgG3 also tended to react relatively less well with gp160 and gp120 and more robustly with gp41 and p24. The contrasting results with serum, as opposed to F105, may result from the polyclonal nature of serum antibodies. There is also a failure to make a robust IgG3 response to neutralizing epitopes on envelope glycoproteins during natural infection. These studies suggest that the investigation of isotype effects on neutralization will require isotype-switched human monoclonal antibodies. Understanding isotype and neutralization will provide important data necessary for designing the most effective possible vaccines.

Naïve and memory B cells in the rhesus macaque can be differentiated by surface expression of CD27 and have differential responses to CD40 ligation.

The rhesus macaque (RM) model has the potential to be an invaluable tool for studying B cell populations during pathogenic infections, however, to date, there has been no definitive delineation of naïve and memory B cell populations in the RM. This has precluded a rigorous analysis of the generation, persistence and resolution of a pathogen-specific memory B cell response. The present study utilized multiple analyses to demonstrate that CD27 expression on B cells is consistent with a memory phenotype. Compared to CD20+CD27- B cells, CD20+CD27+ B cells were larger in size, and preferentially accumulated at effector sites. Direct sequence analysis revealed that CD20+CD27+ B cells had an increased frequency of point mutations that were consistent with somatic hypermutation and at a functional level, CD40 ligation improved CD20+CD27- but not CD20+CD27+ B cell survival in vitro. These data provide definitive evidence that the naïve and memory B cell populations of the RM can be differentiated using surface expression of CD27.

Induction of antibody-mediated neutralization in SIVmac239 by a naturally acquired V3 mutation.

Achieving humoral immunity against human immunodeficiency virus (HIV) is a major obstacle in AIDS vaccine development. Despite eliciting robust humoral responses to HIV, exposed hosts rarely produce broadly neutralizing antibodies. The present study utilizes simian immunodeficiency virus (SIV) to identify viral epitopes that conferred antibody neutralization to clone SIV/17E-CL, an in vivo variant derived from neutralization resistant SIVmac239. Neutralization assays using rhesus macaque monoclonal antibodies were performed on viruses engineered to express single or multiple amino acid mutations. Results identified a single amino acid mutation, P334R, in the carboxy-terminal half of the V3 loop as a critical residue that induced neutralization while retaining normal glycoprotein expression on the surface of the virus. Furthermore, the R334 residue yielded neutralization sensitivity by antibodies recognizing diverse conformational and linear epitopes of gp120, suggesting that neutralization phenotype was a consequence of global structural changes of the envelope protein rather than a specific site epitope.