Detection of antibodies against flavivirus over time in wild non-human primates from the lowlands of Costa Rica.

Two-hundred-nine free ranging non-human primates from 31 locations throughout Costa Rica were captured and released between 1993 and 2012, and blood samples, sera or plasma were collected, to detect antigens and antibodies, and so assess the distribution of active and passive flavivirus infections over time. A competitive enzyme-linked immunoassay for the detection of antibodies was used to determine the distribution of past flavivirus infections over time, while Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was used to detect active West Nile Virus (WNV) and Dengue virus (DENV) infections. The first serological evidence of flavivirus in these animals was determined in 1993, at the same time when DENV re-emerged in humans from Costa Rica. An increase in the number of seropositive wild monkeys to flavivirus was determined over time in the country (11.3% seropositivity in 1993-1996, 20.7% in 2001-2008, and finally 52.9% in 2010-2012). Furthermore, the presence of DENV2 was detected in samples from four howler monkeys collected in 2001-2002, whereas DENV2, DENV3, and DENV4 were found in samples from four white-faced monkeys, and WNV in three howler monkeys living in the Pacific coast of Costa Rica during 2010-2012. The habitat where the positive PCR individuals lived were characterized as fragmented forests, having temperatures ranging from 26°C to 28°C, altitudes below 250 meters above sea level, high precipitation during 7 to 9 months (1500-4000 mm), and a marked dry season of 3 to 5 months. All these animals were living near mangroves; however, they did not show clinical signs of illness at the time of sampling. Results obtained show that the number of seropositive wild non-human primates to flavivirus were increasing during time in the country, longitudinal studies are needed to investigate their role as sentinels of these viruses and to determine if flavivirus infections can affect these species.

Monkey Models and HIV Vaccine Research.

Since the discovery of acquired immunodeficiency syndrome (AIDS) in 1981, it has been extremely difficult to develop an effective vaccine or a therapeutic cure despite over 36 years of global efforts. One of the major reasons is due to the lack of an immune-competent animal model that supports live human immunodeficiency virus (HIV) infection and disease progression such that vaccine-induced correlates of protection and efficacy can be determined clearly before human trials. Nevertheless, rhesus macaques infected with simian immunodeficiency virus (SIV) and chimeric simian human immunodeficiency virus (SHIV) have served as invaluable models not only for understanding AIDS pathogenesis but also for studying HIV vaccine and cure. In this chapter, therefore, we summarize major scientific evidence generated in these models since the beginning of the AIDS pandemic. Hopefully, the accumulated knowledge and lessons contributed by thousands of scientists will be useful in promoting the search of an ultimate solution to end HIV/AIDS.

Novel Monoclonal Antibody LpMab-17 Developed by CasMab Technology Distinguishes Human Podoplanin from Monkey Podoplanin.

Podoplanin (PDPN) is a type-I transmembrane sialoglycoprotein, which possesses a platelet aggregation-stimulating (PLAG) domain in its N-terminus. Among the three PLAG domains, O-glycan on Thr52 of PLAG3 is critical for the binding with C-type lectin-like receptor-2 (CLEC-2) and is essential for platelet-aggregating activity of PDPN. Although many anti-PDPN monoclonal antibodies (mAbs) have been established, almost all mAbs bind to PLAG domains. We recently established CasMab technology to produce mAbs against membranous proteins. Using CasMab technology, we produced a novel anti-PDPN mAb, LpMab-17, which binds to non-PLAG domains. LpMab-17 clearly detected endogenous PDPN of cancer cells and normal cells in Western-blot, flow cytometry, and immunohistochemistry. LpMab-17 recognized glycan-deficient PDPN in flow cytometry, indicating that the interaction between LpMab-17 and PDPN is independent of its glycosylation. The minimum epitope of LpMab-17 was identified as Gly77-Asp82 of PDPN using enzyme-linked immunosorbent assay. Of interest, LpMab-17 did not bind to monkey PDPN, whereas the homology is 94% between human PDPN and monkey PDPN, indicating that the epitope of LpMab-17 is unique compared with the other anti-PDPN mAbs. The combination of different epitope-possessing mAbs could be advantageous for the PDPN-targeting diagnosis or therapy.

Understanding Rift Valley fever: contributions of animal models to disease characterization and control.

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis with devastating health impacts in domestic ruminants and humans. Effective vaccines and accurate disease diagnostic tools are key components in the control of RVF. Animal models reproducing infection with RVF virus are of upmost importance in the development of these disease control tools. Rodent infection models are currently used in the initial steps of vaccine development and for the study of virus induced pathology. Translation of data obtained in these animal models to target species (ruminants and humans) is highly desirable but does not always occur. Small ruminants and non-human primates have been used for pathogenesis and transmission studies, and for testing the efficacy of vaccines and therapeutic antiviral compounds. However, the molecular mechanisms of the immune response elicited by RVF virus infection or vaccination are still poorly understood. The paucity of data in this area offers opportunities for new research activities and programs. This review summarizes our current understanding with respect to immunity and pathogenesis of RVF in animal models with a particular emphasis on small ruminants and non-human primates, including recent experimental infection data in sheep.

Escape from bacterial iron piracy through rapid evolution of transferrin.

Iron sequestration provides an innate defense, termed nutritional immunity, leading pathogens to scavenge iron from hosts. Although the molecular basis of this battle for iron is established, its potential as a force for evolution at host-pathogen interfaces is unknown. We show that the iron transport protein transferrin is engaged in ancient and ongoing evolutionary conflicts with TbpA, a transferrin surface receptor from bacteria. Single substitutions in transferrin at rapidly evolving sites reverse TbpA binding, providing a mechanism to counteract bacterial iron piracy among great apes. Furthermore, the C2 transferrin polymorphism in humans evades TbpA variants from Haemophilus influenzae, revealing a functional basis for standing genetic variation. These findings identify a central role for nutritional immunity in the persistent evolutionary conflicts between primates and bacterial pathogens.

Crucial roles of interleukin-7 in the development of T follicular helper cells and in the induction of humoral immunity.

UNLABELLED: T follicular helper (Tfh) cells are specialized providers of cognate B cell help, which is important in promoting the induction of high-affinity antibody production in germinal centers (GCs). Interleukin-6 (IL-6) and IL-21 have been known to play important roles in Tfh cell differentiation. Here, we demonstrate that IL-7 plays a pivotal role in Tfh generation and GC formation in vivo, as treatment with anti-IL-7 neutralizing antibody markedly impaired the development of Tfh cells and IgG responses. Moreover, codelivery of mouse Fc-fused IL-7 (IL-7-mFc) with a vaccine enhanced the generation of GC B cells as well as Tfh cells but not other lineages of T helper cells, including Th1, Th2, and Th17 cells. Interestingly, a 6-fold-lower dose of an influenza virus vaccine codelivered with Fc-fused IL-7 induced higher antigen-specific and cross-reactive IgG titers than the vaccine alone in both mice and monkeys and led to markedly enhanced protection against heterologous influenza virus challenge in mice. Enhanced generation of Tfh cells by IL-7-mFc treatment was not significantly affected by the neutralization of IL-6 and IL-21, indicating an independent role of IL-7 on Tfh differentiation. Thus, IL-7 holds promise as a critical cytokine for selectively inducing Tfh cell generation and enhancing protective IgG responses. IMPORTANCE: Here, we demonstrate for the first time that codelivery of Fc-fused IL-7 significantly increased influenza virus vaccine-induced antibody responses, accompanied by robust expansion of Tfh cells and GC B cells as well as enhanced GC formation. Furthermore, IL-7-mFc induced earlier and cross-reactive IgG responses, leading to striking protection against heterologous influenza virus challenge. These results suggest that Fc-fused IL-7 could be used for inducing strong and cross-protective humoral immunity against highly mutable viruses, such as HIV and hepatitis C virus, as well as influenza viruses.

Antibody responses to a novel Plasmodium falciparum merozoite surface protein vaccine correlate with protection against experimental malaria infection in Aotus monkeys.

The Block 2 region of the merozoite surface protein-1 (MSP-1) of Plasmodium falciparum has been identified as a target of protective immunity by a combination of seroepidemiology and parasite population genetics. Immunogenicity studies in small animals and Aotus monkeys were used to determine the efficacy of recombinant antigens derived from this region of MSP-1 as a potential vaccine antigen. Aotus lemurinus griseimembra monkeys were immunized three times with a recombinant antigen derived from the Block 2 region of MSP-1 of the monkey-adapted challenge strain, FVO of Plasmodium falciparum, using an adjuvant suitable for use in humans. Immunofluorescent antibody assays (IFA) against erythrocytes infected with P. falciparum using sera from the immunized monkeys showed that the MSP-1 Block 2 antigen induced significant antibody responses to whole malaria parasites. MSP-1 Block 2 antigen-specific enzyme-linked immunosorbent assays (ELISA) showed no significant differences in antibody titers between immunized animals. Immunized animals were challenged with the virulent P. falciparum FVO isolate and monitored for 21 days. Two out of four immunized animals were able to control their parasitaemia during the follow-up period, whereas two out of two controls developed fulminating parasitemia. Parasite-specific serum antibody titers measured by IFA were four-fold higher in protected animals than in unprotected animals. In addition, peptide-based epitope mapping of serum antibodies from immunized Aotus showed distinct differences in epitope specificities between protected and unprotected animals.

A dynamic dose-response model to account for exposure patterns in risk assessment: a case study in inhalation anthrax.

The most commonly used dose-response models implicitly assume that accumulation of dose is a time-independent process where each pathogen has a fixed risk of initiating infection. Immune particle neutralization of pathogens, however, may create strong time dependence; i.e. temporally clustered pathogens have a better chance of overwhelming the immune particles than pathogen exposures that occur at lower levels for longer periods of time. In environmental transmission systems, we expect different routes of transmission to elicit different dose-timing patterns and thus potentially different realizations of risk. We present a dose-response model that captures time dependence in a manner that incorporates the dynamics of initial immune response. We then demonstrate the parameter estimation of our model in a dose-response survival analysis using empirical time-series data of inhalational anthrax in monkeys in which we find slight dose-timing effects. Future dose-response experiments should include varying the time pattern of exposure in addition to varying the total doses delivered. Ultimately, the dynamic dose-response paradigm presented here will improve modelling of environmental transmission systems where different systems have different time patterns of exposure.

Perspectives on clinical and preclinical testing of new tuberculosis vaccines.

This review hopes to improve the selection of new tuberculosis (TB) vaccines by providing several perspectives on the immunization of humans, mice, guinea pigs, rabbits, and monkeys which have not usually been considered. (i) In human TB vaccine trials, the low rate of healing of Mycobacterium bovis BCG lesions (used as the control group) would distinguish individuals who might be helped by vaccination from the 95% who do not need it and would make these trials more conclusive. (ii) The rabbit immune response to Mycobacterium tuberculosis is much more effective in arresting tuberculosis than those of other laboratory animals, so pulmonary tubercle counting in rabbits should be included in all preclinical TB vaccine testing. (iii) Both delayed-type hypersensitivity (DTH) and cell-mediated immunity (CMI) are necessary to control the growth of M. tuberculosis. The testing of new TB vaccines in mice or in guinea pigs may not detect important antigens needed for human immunization. Mice respond poorly to tuberculin-like antigens that cause DTH. Guinea pigs respond poorly to antigens that cause CMI. Rabbits and humans respond well to both DTH and CMI antigens. Since monkeys are very susceptible to M. tuberculosis, they may not be as useful as rabbits for preclinical vaccine evaluation. (iv) Critical antigens (possibly ESAT-6 or CFP-10) might increase the immunity of the host to a greater extent than that produced by a natural M. tuberculosis infection and therefore would be useful in both prophylaxis and immunotherapy. Such critical antigens would increase the host's ability to neutralize key components of M. tuberculosis that enable it to survive in both laboratory animals and humans.

Prospective isolation of mesenchymal stem cells from multiple mammalian species using cross-reacting anti-human monoclonal antibodies.

Mesenchymal stem cells (MSCs) of human and nonhuman mammalian species are often studied for various applications in regenerative medicine research. These MSCs can be derived from human bone marrow (BM) and identified by their ability to form fibroblast-like colony forming units that develop into stromal like cells when expanded in culture. These cells are characterized by their spindle-shaped morphology, their characteristic phenotype (CD73(+), CD90(+), CD105(+), CD45⁻, and CD34⁻), and their ability to differentiate into cells of the osteogenic, adipogenic, and chondrogenic lineages. However, the identification and purification of MSCs from nonhuman mammalian species is hampered by the lack of suitable monoclonal antibodies (mAb). In this report, primary BM and cultured BM-derived MSCs of human and monkey, goat, sheep, dog, and pig were screened for cross-reactivity using a panel of 43 mAb, of which 22 react with either human BM mononuclear cells or cultured human MSCs. We found 7 mAb with specificity for CD271, MSCA-1 (W8B2 antigen), W4A5, CD56, W3C4 (CD349), W5C4, and 58B1, which showed interspecies cross-reactivity. These mAb proved to be useful for prospective sorting of MSCs from the BM of the 6 mammalian species studied as well as for the characterization of their cultured offspring. Flow sorting with the cross-reacting mAb resulted in up to 2400-fold enrichment of the clonogenic cell fraction (fibroblast-like colony forming units). This study provides an important contribution for the comparative prospective isolation of primary BM-MSCs and the characterization of cultured MSCs from multiple mammalian species for preclinical research.

Immunotoxicity testing in nonhuman primates.

Biological relevance is generally the major justification for using nonhuman primates (NHP) during preclinical safety assessment. This holds particularly true for the evaluation of biopharmaceuticals with NHP often being the species of choice. For safety assessment of small molecules, NHP are used in case of a higher degree of metabolic similarity, to detect the highly specific immunotoxic side effects and to discriminate toxicity from efficacy of immunomodulatory drugs. Unlike for rodent immunotoxicity studies, standardized tests and protocols are generally less available for NHP. The immunotoxicity testing protocols described in the present chapter have been adapted for application to NHP samples. In principle, rodent protocols can be transferred to NHP. Fortunately, most of the immunotoxicity parameters delineated in the ICH S8 guideline can be applied to NHP specimens. Exceptions are the host resistance assay and the delayed type hypersensitivity test. Owing to the close structural and physiological similarity between NHP and human, human test kits or reagents are often well suited for application to NHP samples. For data evaluation it should be noted that no inbred strains of NHP are available, resulting in a large inter-animal variability for most immunotoxicity assay results. The experimental protocols and reagents described in this chapter were developed specifically for the cynomolgus monkey (Macaca fascicularis), currently the most commonly used NHP species in toxicology. In many instances, these protocols will also be applicable to rhesus monkeys (M. mulatta) and potentially to other Old World macaques. For the marmoset (Callithrix jacchus), a New World monkey also used in toxicology, the choice of available immunotoxicity testing protocols is much reduced when compared to macaques.

Characterization and antigenicity of the promising vaccine candidate Plasmodium vivax 34kDa rhoptry antigen (Pv34).

This study describes the identification of the Plasmodium vivax rhoptry antigen Pv34 whose sequence was obtained based on homology comparison with the Plasmodium falciparum Pf34. The pv34 gene product was characterized by molecular biology and immunological techniques. Additionally, association of Pv34 to detergent-resistant microdomains (DRMs), expression in late blood-stage parasites and recognition of recombinant Pv34 (rPv34) by sera from P. vivax-infected Aotus monkeys and patients was assessed. Lymphoproliferation and cytokine secretion was also evaluated in individuals living in malaria endemic areas. Altogether, the data support carrying out further studies to assess the immunogenicity and protection-inducing ability of rPv34 as component of a multi-antigenic, multi-stage vaccine against vivax malaria.

Vector-mediated gene transfer engenders long-lived neutralizing activity and protection against SIV infection in monkeys.

The key to an effective HIV vaccine is development of an immunogen that elicits persisting antibodies with broad neutralizing activity against field strains of the virus. Unfortunately, very little progress has been made in finding or designing such immunogens. Using the simian immunodeficiency virus (SIV) model, we have taken a markedly different approach: delivery to muscle of an adeno-associated virus gene transfer vector expressing antibodies or antibody-like immunoadhesins having predetermined SIV specificity. With this approach, SIV-specific molecules are endogenously synthesized in myofibers and passively distributed to the circulatory system. Using such an approach in monkeys, we have now generated long-lasting neutralizing activity in serum and have observed complete protection against intravenous challenge with virulent SIV. In essence, this strategy bypasses the adaptive immune system and holds considerable promise as a unique approach to an effective HIV vaccine.

Localization of West Nile Virus in monkey brain: double staining antigens immunohistochemically of neurons, neuroglia cells and West Nile Virus.

West Nile virus (WNV) can cause encephalitis or meningitis that affects brain tissue, which can also lead to permanent neurological damage that can be fatal. To our knowledge, no consistent double immunohistochemical staining of neurons, neuroglia cells, and WNV has yet been reported. To establish a method for performing double-label immunohistochemical detection of neurons, neuroglia cells and WNV, examining the pathological characteristics of WNV-infected neurons, neuroglia cells, and investigating distribution of WNV in monkey brain, paraffin-embedded monkey brain tissue were retrospectively studied by immunohistochemical staining of neurons, neuroglia cells and WNV. Antibodies against neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP) and WNV were used to develop the method of double-label immunohistochemical staining, which allowed independent assessment of neuron status and WNV distribution. A range of immunohistochemical WNV infection in monkey brain was observed in both neurons and neuroglia cells in terms of the thickness of lesion staining, and the WNV staining was slightly higher in neuroglia cells than in neurons. All these findings suggest that WNV invasion in the brain plays a crucial role in neurological damage by inducing central nervous system (CNS) cell dysfunction or cell death directly.

Inhibition of human sperm motility by contraceptive anti-eppin antibodies from infertile male monkeys: effect on cyclic adenosine monophosphate.

Epididymal protease inhibitor (eppin [official symbol, SPINLW1]) is of interest as a male contraceptive target because of its specificity and location on the human sperm surface. We have examined the effect of anti-eppin antibodies from infertile male monkeys and the effect of recombinant human semenogelin on human sperm motility. Anti-eppin antibodies significantly decreased the progressive motility of human spermatozoa as measured by decreased total distance traveled, decreased straight-line distance, and decreased velocity. Anti-eppin treatment of spermatozoa significantly increased the amount of cAMP present in nonprogressive spermatozoa; however, approximately 25% of antibody-treated spermatozoa could be rescued by the addition of cAMP-acetoxymethyl ester, indicating that anti-eppin-treated spermatozoa have a compromised ability to utilize cAMP. Addition of recombinant human semenogelin has a concentration-dependent inhibitory effect on progressive motility (increased tortuosity and decreased velocity). We tested the hypothesis that anti-eppin antibodies bound to eppin would subsequently block semenogelin binding to eppin. Anti-eppin antibodies from infertile monkeys inhibited eppin from binding to semenogelin. Addition of affinity-purified antibodies made to the dominant C-terminal epitope of eppin had an inhibitory effect on progressive motility (increased tortuosity, decreased velocity, and straight distance). Our results suggest that the eppin-semenogelin binding site is critical for the removal of semenogelin in vivo during semen liquefaction and for the initiation of progressive motility. We conclude that the eppin-semenogelin binding site on the surface of human spermatozoa is an ideal target for a nonsteroidal male contraceptive.

Nosocomial infection of Serratia marcescens may induce a protective effect in monkeys exposed to Bacillus anthracis.

This study was originally designed to collect data on the natural history of inhalational anthrax in a new nonhuman primate model. An uncontrollable event created a new experimental condition which allowed us to retrospectively evaluate the power of the innate immune system to protect from an aerosol exposure of B. anthracis. Five African green monkeys (AGMs) had intravenous catheters implanted. One catheter was accidentally pulled out, leaving four AGMs with catheters and one without. All were exposed, to multiple lethal doses of B. anthracis Ames strain. Blood was collected twice daily to evaluate bacteremia. The AGM with no catheter had blood drawn from a femoral vein and became bacteremic on Day 9; succumbed to inhalational anthrax on Day 10. The other four AGMs had S. marcescens contamination in the catheter; indicated by pure colonies grown from the blood. None of these AGMs showed clinical signs of illness, had B. anthracis or a detectable level of protective antigen in the bloodstream. It appears that the presence of S. marcescens may have induced a "Coley's toxin" effect in this experiment. The innate immune response may have protected the AGMs from a lethal inhalational dose of B. anthracis spores.

Improvement of a recombinant anti-monkey anti-CD3 diphtheria toxin based immunotoxin by yeast display affinity maturation of the scFv.

Recently, a bivalent recombinant anti-human CD3 diphtheria toxin (DT) based immunotoxin derived from the scFv of UCHT1 antibody has been made that shows enhanced bioactivity and is free from the side effects of Fc receptor interaction. In this case, the diminution of CD3 binding due to the placement of the scFv domain at the C-terminus of the truncated DT in single scFv immunotoxins was compensated by adding an additional scFv domain. However, this strategy was less successful for constructing an anti-rhesus recombinant immunotoxin derived from the scFv of FN18 antibody due to poor binding of the anti-rhesus bivalent immunotoxin. We report here that, by increasing the FN18 scFv affinity through random mutagenesis and selection with a dye-labeled monkey CD3epsilongamma recombinant heterodimer, we greatly improved the bioactivity of FN18 derived immunotoxin. The best mutant, C207, contained nine mutations, two of which were located in CDRs that changed the charge from negative to positive. Binding affinity of the C207 scFv to the monkey T cell line HSC-F increased 9.8-fold. The potency of the C207 bivalent immunotoxin assayed by inhibition of protein synthesis increased by 238-fold.