Evaluation of monkeypox- and vaccinia-virus neutralizing antibodies before and after smallpox vaccination: A sero-epidemiological study.

Since May 2022, several countries outside of Africa experienced multiple clusters of monkeypox virus (MPXV)-associated disease. In the present study, anti-MPXV and anti-vaccinia virus (VACV) neutralizing antibody responses were evaluated in two cohorts of subjects from the general Italian population (one half born before the WHO-recommended end of smallpox vaccination in 1980, the other half born after). Higher titers (either against MPXV or VACV) were observed in the cohort of individuals born before the interruption of VACV vaccination. An association between VACV and MPXV antibody levels was observed, suggesting that the smallpox vaccination may confer some degree of cross-protection against MPXV infection. Results from this study highlight low levels of immunity toward the assessed Orthopoxviruses, especially in young adults, advocating the introduction of a VACV- or MPXV-specific vaccine in case of resurgence of monkeypox disease outbreaks.

Longitudinal positron emission tomography and postmortem analysis reveals widespread neuroinflammation in SARS-CoV-2 infected rhesus macaques.

BACKGROUND: Coronavirus disease 2019 (COVID-19) patients initially develop respiratory symptoms, but they may also suffer from neurological symptoms. People with long-lasting effects after acute infections with severe respiratory syndrome coronavirus 2 (SARS-CoV-2), i.e., post-COVID syndrome or long COVID, may experience a variety of neurological manifestations. Although we do not fully understand how SARS-CoV-2 affects the brain, neuroinflammation likely plays a role. METHODS: To investigate neuroinflammatory processes longitudinally after SARS-CoV-2 infection, four experimentally SARS-CoV-2 infected rhesus macaques were monitored for 7 weeks with 18-kDa translocator protein (TSPO) positron emission tomography (PET) using [18F]DPA714, together with computed tomography (CT). The baseline scan was compared to weekly PET-CTs obtained post-infection (pi). Brain tissue was collected following euthanasia (50 days pi) to correlate the PET signal with TSPO expression, and glial and endothelial cell markers. Expression of these markers was compared to brain tissue from uninfected animals of comparable age, allowing the examination of the contribution of these cells to the neuroinflammatory response following SARS-CoV-2 infection. RESULTS: TSPO PET revealed an increased tracer uptake throughout the brain of all infected animals already from the first scan obtained post-infection (day 2), which increased to approximately twofold until day 30 pi. Postmortem immunohistochemical analysis of the hippocampus and pons showed TSPO expression in cells expressing ionized calcium-binding adaptor molecule 1 (IBA1), glial fibrillary acidic protein (GFAP), and collagen IV. In the hippocampus of SARS-CoV-2 infected animals the TSPO+ area and number of TSPO+ cells were significantly increased compared to control animals. This increase was not cell type specific, since both the number of IBA1+TSPO+ and GFAP+TSPO+ cells was increased, as well as the TSPO+ area within collagen IV+ blood vessels. CONCLUSIONS: This study manifests [18F]DPA714 as a powerful radiotracer to visualize SARS-CoV-2 induced neuroinflammation. The increased uptake of [18F]DPA714 over time implies an active neuroinflammatory response following SARS-CoV-2 infection. This inflammatory signal coincides with an increased number of TSPO expressing cells, including glial and endothelial cells, suggesting neuroinflammation and vascular dysregulation. These results demonstrate the long-term neuroinflammatory response following a mild SARS-CoV-2 infection, which potentially precedes long-lasting neurological symptoms.

Immunity to measles in Italian children and adolescents: a persistent problem in view of measles elimination.

BACKGROUND: Despite efforts to increase coverage by two doses of measles vaccine in Italy, measles continues to circulate, with over 13 000 cases of disease since 2013. This study aimed to evaluate immunity to measles in Italian children and adolescents. METHODS: A total of 378 serum samples from subjects aged 9 months-18 years were collected in Northern, Central and Southern regions of Italy between 2012 and 2016. Specific IgG antibodies against measles were measured by a commercial ELISA kit. RESULTS: The frequency of IgG-positive samples ranged from 10.5% in infants under 1 year to 98.3% in children aged 6-7 years. The frequency of IgG was 72.2% in subjects aged 1-2 years, 85.6% in those aged 3-5 years and 88.3 and 86.8% in those aged 8-10 and 11-18 years, respectively. In Northern Italy, IgG prevalence was consistent with data on vaccination coverage, whereas some differences were observed in samples from subjects aged more than 8 years in Central and Southern Italy. CONCLUSIONS: Our findings confirm that a large proportion of children and adolescents in Italy are still susceptible to measles. While data on first- and second-dose measles vaccination are essential, they are not sufficient to identify susceptible population cohorts to be targeted by vaccination.

Identification of adjuvants for clinical trials performed with Plasmodium falciparum AMA1 in rabbits.

BACKGROUND: In this study, seven adjuvants were compared for use with Plasmodium falciparum DiCo-Apical Membrane Antigen 1 (Pf-DiCo-AMA1), with the aim to identify an ideal adjuvant which yields high antibody titres and potentially broadens the responses in clinical trials. The following adjuvant formulations were evaluated: SE, SE-GLA, Liposomes, Liposomes-GLA, CoVaccine HT™, ImSaVac-P and ImSaVac-P o/w. The study was performed in rabbits, which were immunized with FVO-AMA1 in combination with one of the seven adjuvants. Antibody levels (humoral responses) and functional activity of the antibodies induced against malaria vaccine candidate AMA1 were evaluated. Thus, in this study the ideal adjuvant is expected to induce high functional antibody levels, a long-lived response, and a broad cross-strain activity. RESULTS: AMA1 formulated in all adjuvants was immunogenic. However, the magnitude of the immune responses differed between the seven adjuvants. The highest IgG levels were observed for the CoVaccine HT™ group, this was statistically significant for all four AMA1 variants versus all other adjuvant groups. No differences were observed in the breadth of the humoral response, i.e., increased recognition of AMA1 variants. Also, Growth Inhibition Activity (GIA) for both Plasmodium falciparum strains (FCR3 - homologous to FVO AMA1 protein and NF54 - heterologous to FVO AMA1 protein) were significantly higher in the CoVaccine HT™ group as compared to the other adjuvant groups. CONCLUSIONS: In brief, all seven vaccine - adjuvant formulations were immunogenic. The magnitude of the immune responses differed between the seven adjuvants. No statistically significant differences were observed in the breadth of the humoral response, nor in longevity of the response. Nevertheless, AMA1 formulated in CoVaccine HT™ appeared as the best adjuvant for use in clinical trials.

Influenza virus infection as well as immunization with DNA encoding haemagglutinin protein induces potent antibody-dependent phagocytosis (ADP) and monocyte infection-enhancing responses in macaques.

Antibodies directed against the conserved regions within the influenza A haemagglutinin (HA) protein are detected at low frequency in humans. These antibodies display a broad reactivity against divergent influenza virus strains and could potentially offer broad protection. The in vivo protective effect of these antibodies is mainly mediated through Fc receptor effector functions. While antibody-dependent phagocytosis (ADP) of anti-HA antibodies has been demonstrated in human sera and sera from influenza virus-infected macaques, it is not known whether ADP can also be induced by vaccination and what the relative strength of ADP responses is in comparison to other antibody functions. Using a cohort of influenza virus-infected and immunized macaques, we demonstrate that infection as well as immunization with DNA-encoding HA induces high-titre ADP responses against HA, which are of potency 100-1000 times higher than virus inhibitory functions including antibody-dependent cell-mediated cytotoxicity (ADCC), virus neutralization (VN) and haemagglutinin inhibition (HAI). ADP activity was equally high against HA of heterologous influenza strains of the same subtype, in contrast to virus inhibitory functions, which were all greatly diminished. ADP titres against H5, representing a hetero-subtypic virus, were much lower. ADP was measured in THP-1 cells but was also observed in primary peripheral blood monocytes and neutrophils. Furthermore, at high serum dilution enhanced infection of both monocytes and myeloid dendritic cells (mDC) was observed. Hence, influenza virus infection as well as DNA-immunization against HA can induce high-titre ADP responses that can potentially enhance influenza virus infection of primary phagocytic and dendritic cells.

Plasmodium falciparum merozoite surface antigen-specific cytophilic IgG and control of malaria infection in a Beninese birth cohort.

BACKGROUND: Substantial evidence indicates that cytophilic IgG responses to Plasmodium falciparum merozoite antigens play a role in protection from malaria. The specific targets mediating immunity remain unclear. Evaluating antibody responses in infants naturally-exposed to malaria will allow to better understand the establishment of anti-malarial immunity and to contribute to a vaccine development by identifying the most appropriate merozoite candidate antigens. METHODS: The study was based on parasitological and clinical active follow-up of infants from birth to 18 months of age conducted in the Tori Bossito area of southern Benin. For 399 infants, plasma levels of cytophilic IgG antibodies with specificity for five asexual stage malaria vaccine candidate antigens were determined by ELISA in infants' peripheral blood at 6, 9, 12 and 15 months of age. Multivariate mixed logistic model was used to investigate the association between antibody levels and anti-malarial protection in the trimester following the IgG quantification. Moreover, the concentrations of merozoite antigen-specific IgG were compared between a group of infants apparently able to control asymptomatic malaria infection (CAIG) and a group of infants with no control of malaria infection (Control group (NCIG)). Protective effect of antibodies was also assessed after 15 months of malaria exposure with a Cox regression model adjusted on environmental risk. RESULTS: Cytophilic IgG responses to AMA1, MSP1, MSP2-3D7, MSP2-FC27, MSP3 and GLURP R2 were associated with increasing malarial infection risk in univariate analysis. The multivariate mixed model showed that IgG1 and IgG3 to AMA1 were associated with an increased risk of malarial infection. However infants from CAIG (n = 53) had significantly higher AMA1-, MSP2-FC27-, MSP3-specific IgG1 and AMA1-, MSP1-, MSP2-FC27-, MSP3 and GLURP-R2-specific IgG3 than those from NCIG (n = 183). The latter IgG responses were not associated with protection against clinical malaria in the whole cohort when protective effect is assessed after 15 months of malaria exposition. CONCLUSION: In this cohort, merozoite antigen-specific cytophilic IgG levels represent a marker of malaria exposure in infants from 6 to 18 months of age. However, infants with resolution of asymptomatic infection (CAIG) seem to have acquired naturally immunity against P. falciparum. This observation is encouraging in the context of the development of multitarget P. falciparum vaccines.

Defining predictive factors for reproductive output in captive common marmosets (Callithrix jacchus).

Common marmosets (Callithrix jacchus) demonstrate variations in reproductive output, not only in terms of total reproductive output during a lifetime but also in litter size per parturition. The present study explores factors, such as parents' litter size, parturition number, maternal body weight at conception and maternal age, which may account for this variation. A retrospective analysis of clinical records of a captive breeding colony was conducted over a 9-year period yielding reproductive summaries of 26 dams and 22 sires producing a total of 115 litters. Dams born from litters of ≤2 (N = 20) more often produced litters of ≤2, whereas dams born from litters of >2 (N = 6) more often produced litters of >2 (p < 0.05). The dams' maternal body weight at the time of conception had also a significant effect on subsequent litter size. In addition, the chance of triplets was higher after the second parturition. Maternal age, interbirth interval, and season of birth had no effect on litter size. Factors relating to the sire had a negligible effect on the size of the litter. Multivariate statistical modeling revealed that the dams' original litter size, maternal bodyweight at conception and parturition number are determining factors for the number of babies per litter. This study identified factors determining marmoset litter size, some of which (maternal litter size) are novel to this study and were not reported previously. Further exploration of the potential role of maternal litter size as a determinant of the litter sizes produced by marmoset breeders is warranted.

Effects of buprenorphine, butorphanol or tramadol premedication on anaesthetic induction with alfaxalone in common marmosets (Callithrix jacchus).

OBJECTIVE: To investigate the clinical and physiological effects of intravenous (IV) alfaxalone alone or in combination with buprenorphine, butorphanol or tramadol premedication in marmosets. STUDY DESIGN: Prospective, randomized, blinded, crossover design. ANIMALS: Nine healthy marmosets (391 ± 48 g, 3.7 ± 2.2 years old). METHODS: Meloxicam 0.20 mg kg-1 subcutaneously, atropine 0.05 mg kg-1 intramuscularly (IM) and either buprenorphine 20 µg kg-1 IM (BUP-A), butorphanol 0.2 mg kg-1 IM (BUT-A), tramadol 1.5 mg kg-1 IM (TRA-A) or no additional drug (control) were administered to all marmosets as premedication. After 1 hour, anaesthesia was induced with 16 mg kg-1 alfaxalone IV. All animals received all protocols. The order of protocol allocation was randomized with a minimum 28 day wash-out period. During anaesthesia, respiratory and pulse rates, rectal temperature, haemoglobin oxygen saturation, arterial blood pressure, palpebral and pedal withdrawal reflexes and degree of muscle relaxation were assessed and recorded every 5 minutes. Quality of induction and recovery were assessed. Duration of induction, immobilization and recovery were recorded. Blood samples were analysed for aspartate aminotransferase, creatine kinase and lactate dehydrogenase concentrations. The protocols were compared using paired t tests, Wilcoxon's signed-rank test with Bonferroni's corrections and linear mixed effect models where appropriate. RESULTS: Out of nine animals, apnoea was noted in eight animals administered protocol BUP-A and two animals administered protocol BUT-A. With TRA-A and control protocols, apnoea was not observed. No other significant differences in any of the parameters were found; however, low arterial blood pressures and hypoxia occurred in TRA-A. CONCLUSIONS AND CLINICAL RELEVANCE: Our study employing different premedications suggests that the previously published dose of 16 mg kg-1 alfaxalone is too high when used with premedication because we found a high incidence of complications including apnoea (BUP-A), hypotension and hypoxaemia (TRA-A). Appropriate monitoring and countermeasures are recommended.

Correlation between Virus Replication and Antibody Responses in Macaques following Infection with Pandemic Influenza A Virus.

UNLABELLED: Influenza virus infection of nonhuman primates is a well-established animal model for studying pathogenesis and for evaluating prophylactic and therapeutic intervention strategies. However, usually a standard dose is used for the infection, and there is no information on the relation between challenge dose and virus replication or the induction of immune responses. Such information is also very scarce for humans and largely confined to evaluation of attenuated virus strains. Here, we have compared the effect of a commonly used dose (4 × 10(6) 50% tissue culture infective doses) versus a 100-fold-higher dose, administered by intrabronchial installation, to two groups of 6 cynomolgus macaques. Animals infected with the high virus dose showed more fever and had higher peak levels of gamma interferon in the blood. However, virus replication in the trachea was not significantly different between the groups, although in 2 out of 6 animals from the high-dose group it was present at higher levels and for a longer duration. The virus-specific antibody response was not significantly different between the groups. However, antibody enzyme-linked immunosorbent assay, virus neutralization, and hemagglutination inhibition antibody titers correlated with cumulative virus production in the trachea. In conclusion, using influenza virus infection in cynomolgus macaques as a model, we demonstrated a relationship between the level of virus production upon infection and induction of functional antibody responses against the virus. IMPORTANCE: There is only very limited information on the effect of virus inoculation dose on the level of virus production and the induction of adaptive immune responses in humans or nonhuman primates. We found only a marginal and variable effect of virus dose on virus production in the trachea but a significant effect on body temperature. The induction of functional antibody responses, including virus neutralization titer, hemagglutination inhibition titer, and antibody-dependent cell-mediated cytotoxicity, correlated with the level of virus replication measured in the trachea. The study reveals a relationship between virus production and functional antibody formation, which could be relevant in defining appropriate criteria for new influenza virus vaccine candidates.

Protection against malaria after immunization by chloroquine prophylaxis and sporozoites is mediated by preerythrocytic immunity.

Volunteers immunized under chloroquine chemoprophylaxis with Plasmodium falciparum sporozoites (CPS) develop complete, long-lasting protection against homologous sporozoite challenge. Chloroquine affects neither sporozoites nor liver-stages, but kills only asexual forms in erythrocytes once released from the liver into the circulation. Consequently, CPS immunization exposes the host to antigens from both preerythrocytic and blood stages, and induced immunity might target either of these stages. We therefore explored the life cycle stage specificity of CPS-induced protection. Twenty-five malaria-naïve volunteers were enrolled in a clinical trial, 15 of whom received CPS immunization. Five immunized subjects and five controls received a sporozoite challenge by mosquito bites, whereas nine immunized and five control subjects received an i.v. challenge with P. falciparum-infected erythrocytes. The latter approach completely bypasses preerythrocytic stages, enabling a direct comparison of protection against either life cycle stage. CPS-immunized subjects (13 of 14) developed anticircumsporozoite antibodies, whereas only one volunteer generated minimal titers against typical blood-stage antigens. IgG from CPS-immunized volunteers did not inhibit asexual blood-stage growth in vitro. All CPS-immunized subjects (5 of 5) were protected against sporozoite challenge. In contrast, nine of nine CPS-immunized subjects developed parasitemia after blood-stage challenge, with identical prepatent periods and blood-stage multiplication rates compared with controls. Intravenously challenged CPS-immunized subjects showed earlier fever and increased plasma concentrations of inflammatory markers D-dimer, IFN-γ, and monokine induced by IFN-γ than i.v. challenged controls. The complete lack of protection against blood-stage challenge indicates that CPS-induced protection is mediated by immunity against preerythrocytic stages. However, evidence is presented for immune recognition of P. falciparum-infected erythrocytes, suggesting memory responses unable to generate functional immunity.

KAI407, a potent non-8-aminoquinoline compound that kills Plasmodium cynomolgi early dormant liver stage parasites in vitro.

Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, and exoerythrocytic development is monitored in the presence of compounds. Liver stage cultures are fixed after 6 days and stained with anti-Hsp70 antibodies, and the relative proportions of small (hypnozoite) and large (schizont) forms relative to the untreated controls are determined. This assay was used to screen a series of 18 known antimalarials and 14 new non-8-aminoquinolines (preselected for blood and/or liver stage activity) in three-point 10-fold dilutions (0.1, 1, and 10 µM final concentrations). A novel compound, designated KAI407 showed an activity profile similar to that of primaquine (PQ), efficiently killing the earliest stages of the parasites that become either primary hepatic schizonts or hypnozoites (50% inhibitory concentration [IC50] for hypnozoites, KAI407, 0.69 µM, and PQ, 0.84 µM; for developing liver stages, KAI407, 0.64 µM, and PQ, 0.37 µM). When given as causal prophylaxis, a single oral dose of 100 mg/kg of body weight prevented blood stage parasitemia in mice. From these results, we conclude that KAI407 may represent a new compound class for P. vivax malaria prophylaxis and potentially a radical cure.

Hematological and Serum Biochemical Reference Intervals for Alphaxalone Sedated Common Marmosets (Callithrix jacchus).

Marmosets are routinely used in biomedical research, therefore there is an increasing need for updated reference intervals calculated using a large sample size, correct statistics, and considering different variables. Hematological and biochemical values from 472 healthy common marmosets sedated with alphaxalone were collected over a ten-year period (2013-2023). The variables assumed to have influenced the blood-based parameters were compared, i.e., sex, age, housing condition, pregnancy, and contraceptive use. Reference intervals were calculated based on observed percentiles without parametric assumptions, and with parametric assumptions following Box-Cox transformation. Juvenile marmosets showed increased ALP, phosphate, WBC, lymphocyte count, and basophil count and decreased levels of GGT and Fe compared to adults. Marmosets housed strictly indoors showed increased ALT and GGT levels and decreased levels of total bilirubin and neutrophil count compared to marmosets housed with outdoor access. Pregnant marmosets showed increased ALP, total bilirubin, neutrophil count, monocyte count, and basophil count, and decreased levels of AST, ALT, cholesterol, Fe, and lymphocyte count compared to non-pregnant marmosets. Etonogestrel contracepted marmosets showed decreased P-LCR compared to females who were not contracepted. Updated reference intervals will aid researchers and veterinarians in identifying physiological and pathological changes, as well as improve the reproducibility of research in this species.

A Diversity Covering (DiCo) Plasmodium vivax apical membrane antigen-1 vaccine adjuvanted with RFASE/RSL10 yields high levels of growth-inhibitory antibodies.

Plasmodium vivax malaria is increasingly recognized as a major global health problem and the socio-economic impact of P.vivax-induced burden is huge. Vaccine development against P. vivax malaria has been hampered by the lack of an in vitro culture system and poor access to P. vivax sporozoites. The recent generation of Plasmodium falciparum parasites that express a functional P. vivax AMA1 molecule has provided a platform for in vitro evaluation of PvAMA1 as a potential blood stage vaccine. Three so-called PvAMA1 Diversity Covering (DiCo) proteins were designed to assess their potential to induce a functional and broad humoral immune response to the polymorphic PvAMA1 molecule. Rabbits were immunized with the mixture of three, Pichia-produced, PvAMA1 DiCo proteins, as well as with 2 naturally occurring PvAMA1 alleles. For these three groups, the experimental adjuvant raffinose fatty acid sulfate ester (RFASE) was used, while in a fourth group the purified main mono-esterified constituent (RSL10) of this adjuvant was used. Animals immunized with the mixture of the three PvAMA1 DiCo proteins in RFASE showed high anti-PvAMA1 antibody titers against three naturally occurring PvAMA1variants while also high growth-inhibitory capacity was observed against P. falciparum parasites expressing PvAMA1. This supports further clinical development of the PvAMA1 DiCo mixture as a potential malaria vaccine. However, as the single allele PvAMA1 SalI-group showed similar characteristics in antibody titer and inhibition levels as the PvAMA1 DiCo mixture-group, this raises the question whether a mixture is really necessary to overcome the polymorphism in the vaccine candidate. RFASE induced strong humoral responses, as did the animals immunized with the purified component, RSL10. This suggests that RSL10 is the active ingredient. However, one of the RSL10-immunized animal showed a delayed response, necessitating further research into the clinical development of RSL10.

Plasmodium falciparum AMA1 and CSP antigen diversity in parasite isolates from southern Ghana.

Introduction: Diversity in malarial antigens is an immune evasion mechanism that gives malaria parasites an edge over the host. Immune responses against one variant of a polymorphic antigen are usually not fully effective against other variants due to altered epitopes. This study aimed to evaluate diversity in the Plasmodium falciparum antigens apical membrane antigen 1 (PfAMA1) and circumsporozoite protein (PfCSP) from circulating parasites in a malaria-endemic community in southern Ghana and to determine the effects of polymorphisms on antibody response specificity. Methods: The study involved 300 subjects, whose P. falciparum infection status was determined by microscopy and PCR. Diversity within the two antigens was evaluated by msp2 gene typing and molecular gene sequencing, while the host plasma levels of antibodies against PfAMA1, PfCSP, and two synthetic 24mer peptides from the conserved central repeat region of PfCSP, were measured by ELISA. Results: Of the 300 subjects, 171 (57%) had P. falciparum infection, with 165 of the 171 (96.5%) being positive for either or both of the msp2 allelic families. Gene sequencing of DNA from 55 clonally infected samples identified a total of 56 non-synonymous single nucleotide polymorphisms (SNPs) for the Pfama1 gene and these resulted in 44 polymorphic positions, including two novel positions (363 and 365). Sequencing of the Pfcsp gene from 69 clonal DNA samples identified 50 non-synonymous SNPs that resulted in 42 polymorphic positions, with half (21) of these polymorphic positions being novel. Of the measured antibodies, only anti-PfCSP antibodies varied considerably between PCR parasite-positive and parasite-negative persons. Discussion: These data confirm the presence of a considerable amount of unique, previously unreported amino acid changes, especially within PfCSP. Drivers for this diversity in the Pfcsp gene do not immediately seem apparent, as immune pressure will be expected to drive a similar level of diversity in the Pfama1 gene.

Diversity covering AMA1-MSP119 fusion proteins as malaria vaccines.

To overcome polymorphism in the malaria vaccine candidate Plasmodium falciparum apical membrane antigen 1 (PfAMA1), fusion protein chimeras comprised of three diversity-covering (DiCo) PfAMA1 molecules (D1, D2, and D3) and two allelic variants of the C-terminal 19-kDa region of merozoite surface protein 1 (MSP119) (variants M1 and M2) were generated. A mixture of fusion proteins (D1M1/D2M2D3) and the D1M1D2M2D3 fusion were compared to a single-unit mixture (D1/D2/D3/M1) in an immunological study in groups of rabbits. Following immunization, titers of antibodies (Abs) against four naturally occurring PfAMA1 alleles were high for all groups, as were growth inhibition assay (GIA) levels against two antigenically distinct laboratory parasite strains. Fusion of AMA1 to MSP119 did not suppress levels of antibodies against the AMA1 component. In addition, the breadth of antibody responses was unaffected. Anti-AMA1 antibodies were largely responsible for parasite growth inhibition, as shown in reversal-of-inhibition experiments by adding competing AMA1 antigen. For all groups, titration of the MSP119 antigen into the GIA led to only a small decrease in parasite inhibition, although titers of antibodies against MSP119 were increased 15-fold for the groups immunized with fusion proteins. GIA with affinity-purified anti-MSP119 antibodies showed that the 50% inhibitory concentrations of the anti-MSP119 antibody preparations were in the same order of magnitude for all animals tested, leading to the conclusion that fusing MSP119 to PfAMA1 leads to a small but significant increase in functional antibody levels. This study shows that combination of multiple vaccine candidates in fusion proteins may lead to improved characteristics of the vaccine.

Haplotype diversity generated by ancient recombination-like events in the MHC of Indian rhesus macaques.

The Mamu-A, Mamu-B, and Mamu-DRB genes of the rhesus macaque show several levels of complexity such as allelic heterogeneity (polymorphism), copy number variation, differential segregation of genes/alleles present on a haplotype (diversity) and transcription level differences. A combination of techniques was implemented to screen a large panel of pedigreed Indian rhesus macaques (1,384 individuals representing the offspring of 137 founding animals) for haplotype diversity in an efficient and inexpensive manner. This approach allowed the definition of 140 haplotypes that display a relatively low degree of region variation as reflected by the presence of only 17 A, 18 B and 22 DRB types, respectively, exhibiting a global linkage disequilibrium comparable to that in humans. This finding contrasts with the situation observed in rhesus macaques from other geographic origins and in cynomolgus monkeys from Indonesia. In these latter populations, nearly every haplotype appears to be characterised by a unique A, B and DRB region. In the Indian population, however, a reshuffling of existing segments generated "new" haplotypes. Since the recombination frequency within the core MHC of the Indian rhesus macaques is relatively low, the various haplotypes were most probably produced by recombination events that accumulated over a long evolutionary time span. This idea is in accord with the notion that Indian rhesus macaques experienced a severe reduction in population during the Pleistocene due to a bottleneck caused by geographic changes. Thus, recombination-like processes appear to be a way to expand a diminished genetic repertoire in an isolated and relatively small founder population.

Comparison of three different sedative-anaesthetic protocols (ketamine, ketamine-medetomidine and alphaxalone) in common marmosets (Callithrix jacchus).

BACKGROUND: Handling of common marmoset (Callithrix jacchus) usually requires chemical restraint. Ketamine has been associated with muscle damage in primates, while common marmosets, compared to other primates, additionally display an exceptional high sensitivity to ketamine-associated side-effects. Notably, muscle twitching movements of limbs and hands, and a marked increase in salivation are observed. We investigated two alternative intramuscular (i.m.) immobilisation protocols against ketamine (50 mg/kg; protocol 1) in a double-blind randomised crossover study in ten healthy adult common marmosets for use as a safe reliable, short-term immobilisation and sedation. These protocols comprised: alphaxalone (12 mg/kg; protocol 2) and 25 mg/kg ketamine combined with 0.50 mg/kg medetomidine (reversal with 2.5 mg/kg atipamezole; protocol 3A). Following completion and unblinding, the project was extended with an additional protocol (3B), comprising 25 mg/kg ketamine combined with 0.05 mg/kg medetomidine (reversal with 0.25 mg/kg atipamezole, twice with 35 min interval). RESULTS: All protocols in this study provided rapid onset (induction times <5 min) of immobilisation and sedation. Duration of immobilisation was 31.23 ± 22.39 min, 53.72 ± 13.08 min, 19.73 ± 5.74 min, and 22.78 ± 22.37 min for protocol 1, 2, 3A, and 3B, respectively. Recovery times were 135.84 ± 39.19 min, 55.79 ± 11.02 min, 405.46 ± 29.81 min, and 291.91 ± 80.34 min, respectively. Regarding the quality, and reliability (judged by pedal withdrawal reflex, palpebral reflex and muscle tension) of all protocols, protocol 2 was the most optimal. Monitored vital parameters were within clinically acceptable limits during all protocols and there were no fatalities. Indication of muscle damage as assessed by AST, LDH and CK values was most prominent elevated in protocol 1, 3A, and 3B. CONCLUSIONS: We conclude that intramuscular administration of 12 mg/kg alphaxalone to common marmosets is preferred over other protocols studied. Protocol 2 resulted in at least comparable immobilisation quality with acceptable and less frequent side effects and superior recovery quality. In all protocols, supportive therapy, such as external heat support, remains mandatory. Notably, an unacceptable long recovery period in both ketamine/medetomidine protocols (subsequently reversed with atipamezole) was observed, showing that α-2 adrenoreceptor agonists in the used dose and dosing regime is not the first choice for sedation in common marmosets in a standard research setting.

AIDS-protective HLA-B*27/B*57 and chimpanzee MHC class I molecules target analogous conserved areas of HIV-1/SIVcpz.

In the absence of treatment, most HIV-1-infected humans develop AIDS. However, a minority are long-term nonprogressors, and resistance is associated with the presence of particular HLA-B*27/B*57 molecules. In contrast, most HIV-1-infected chimpanzees do not contract AIDS. In comparison with humans, chimpanzees experienced an ancient selective sweep affecting the MHC class I repertoire. We have determined the peptide-binding properties of frequent chimpanzee MHC class I molecules, and show that, like HLA-B*27/B*57, they target similar conserved areas of HIV-1/SIV(cpz). In addition, many animals appear to possess multiple molecules targeting various conserved areas of the HIV-1/SIV(cpz) Gag protein, a quantitative aspect of the immune response that may further minimize the chance of viral escape. The functional characteristics of the contemporary chimpanzee MHC repertoire suggest that the selective sweep was caused by a lentiviral pandemic.

Evaluation of Anesthetic and Cardiorespiratory Effects after Intramuscular Administration of Three Different Doses of Telazol® in Common Marmosets (Callithrix jacchus).

Marmosets' small body size makes anesthesia challenging. Ideally, small volumes of drugs should be administered intramuscularly (i.m.). In addition, dose-dependent sedation and anesthesia are desirable properties for sedatives and anesthetics in marmosets. Telazol® (tiletamine and zolazepam) is highly concentrated, allowing the use of small injection volumes and dose-dependent sedation and anesthesia. A randomized, blinded study with crossover design in ten healthy adult common marmosets (Callithrix jacchus) was performed to evaluate the anesthetic and cardiorespiratory effects of three doses of i.m. Telazol® (respectively, 5, 10, and 15 mg/kg). Depth of anesthesia, cardiorespiratory effects, and induction, immobilization, and recovery times were determined. A significant difference was observed in immobilization time between 5 and 15 mg/kg of Telazol®. In addition, 15 mg/kg of Telazol® resulted in increased recovery times compared to 5 mg/kg. The cardiorespiratory effects during the first 45 min of immobilization were within clinically acceptable limits. The pedal withdrawal reflex was the best indicator of the anesthetic depth.

Reference Intervals and Percentiles for Hematologic and Serum Biochemical Values in Captive Bred Rhesus (Macaca mulatta) and Cynomolgus Macaques (Macaca fascicularis).

Several physiological characteristics and housing conditions are known to affect hematologic and serum biochemical values in macaques. However, the studies that have been conducted either report values calculated based on a small number of animals, were designed specifically to document the effect of a particular condition on the normal range of hematologic and serum biochemical values, or used parametric assumptions to calculate hematologic and serum biochemical reference intervals. We conducted a retrospective longitudinal cohort study to estimate reference intervals for hematologic and serum biochemical values in clinically healthy macaques based on observed percentiles without parametric assumptions. Data were obtained as part of the Biomedical Primate Research Centre (Rijswijk, The Netherlands) health monitoring program between 2018 and 2021. In total, 4009 blood samples from 1475 macaques were analyzed with a maximum of one repeat per year per animal. Data were established by species, gender, age, weight-for-height indices, pregnancy, sedation protocol, and housing conditions. Most of the parameters profoundly affected just some hematologic and serum biochemical values. A significant glucose difference was observed between the ketamine and ketamine-medetomidine sedation protocols. The results emphasize the importance of establishing uniform experimental groups with validated animal husbandry and housing conditions to improve the reproducibility of the experiments.