Divergent evolution of protein conformational dynamics in dihydrofolate reductase.

Molecular evolution is driven by mutations, which may affect the fitness of an organism and are then subject to natural selection or genetic drift. Analysis of primary protein sequences and tertiary structures has yielded valuable insights into the evolution of protein function, but little is known about the evolution of functional mechanisms, protein dynamics and conformational plasticity essential for activity. We characterized the atomic-level motions across divergent members of the dihydrofolate reductase (DHFR) family. Despite structural similarity, Escherichia coli and human DHFRs use different dynamic mechanisms to perform the same function, and human DHFR cannot complement DHFR-deficient E. coli cells. Identification of the primary-sequence determinants of flexibility in DHFRs from several species allowed us to propose a likely scenario for the evolution of functionally important DHFR dynamics following a pattern of divergent evolution that is tuned by cellular environment

Transfer of maternal immunity and programming of the newborn immune system

As placental mammals, the pregnant women and the fetus have intense and prolonged interactions during gestation. There is increasing evidence that multiple molecular as well as cellular components originating in pregnant women are transferred to the fetus. The transfer of maternal antibodies has long been recognized as a central component of newborn immunity against pathogens. More recent studies indicate that inflammatory mediators, micronutrients, microbial products and maternal cells are transferred in utero and influence the fetal immune system. Together, these multiple signals are likely to form a complex network of interactions that program the neonatal immune system and tune its homeostatic regulation. Maternal disorders, in particular infectious diseases, modify these signals and may thereby alter immunity in early life. Understanding maternal programming of the newborn immune system could provide a basis for interventions promoting child health.SCOPUS: re.jinfo:eu-repo/semantics/publishe

IgG3 collaborates with IgG1 and IgA to recruit effector function in RV144 vaccinees

While the RV144 HIV vaccine trial led to moderately reduced risk of HIV acquisition, emerging data from the HVTN702 trial point to the critical need to reexamine RV144-based correlates of reduced risk of protection. While in RV144, the induction of V2-binding, non-IgA, IgG3 antibody responses with nonneutralizing functions were linked to reduced risk of infection, the interactions between these signatures remain unclear. Thus, here we comprehensively profile the humoral immune response in 300 RV144 vaccinees to decipher the relationships between humoral biomarkers of protection. We found that vaccine-specific IgG1, IgG3, and IgA were highly correlated. However, ratios of IgG1:IgG3:IgA provided insights into subclass/isotype polyclonal functional regulation. For instance, in the absence of high IgG1 levels, IgG3 antibodies exhibited limited functional activity, pointing to IgG3 as a critical contributor, but not sole driver, of effective antiviral humoral immunity. Higher IgA levels were linked to enhanced antibody effector function, including neutrophil phagocytosis (ADNP), complement deposition (ADCD), and antibody-dependent NK degranulation (CD107a), some of which were increased in infected vaccinees in a case/control data set, suggesting that IgA-driven functions compromised immunity. These data highlight the interplay between IgG1, IgG3, and IgA, pointing to the need to profile the relationships between subclass/isotype selection

Vectored delivery of anti-SIV envelope targeting mAb via AAV8 protects rhesus macaques from repeated limiting dose intrarectal swarm SIVsmE660 challenge.

Gene based delivery of immunoglobulins promises to safely and durably provide protective immunity to individuals at risk of acquiring infectious diseases such as HIV. We used a rhesus macaque animal model to optimize delivery of naturally-arising, autologous anti-SIV neutralizing antibodies expressed by Adeno-Associated Virus 8 (AAV8) vectors. Vectored transgene expression was confirmed by quantitation of target antibody abundance in serum and mucosal surfaces. We tested the expression achieved at varying doses and numbers of injections. Expression of the transgene reached a saturation at about 2 x 10(12) AAV8 genome copies (gc) per needle-injection, a physical limitation that may not scale clinically into human trials. In contrast, expression increased proportionately with the number of injections. In terms of anti-drug immunity, anti-vector antibody responses were universally strong, while those directed against the natural transgene mAb were detected in only 20% of animals. An anti-transgene antibody response was invariably associated with loss of detectable plasma expression of the antibody. Despite having atypical glycosylation profiles, transgenes derived from AAV-directed muscle cell expression retained full functional activity, including mucosal accumulation, in vitro neutralization, and protection against repeated limiting dose SIVsmE660 swarm challenge. Our findings demonstrate feasibility of a gene therapy-based passive immunization strategy against infectious disease, and illustrate the potential for the nonhuman primate model to inform clinical AAV-based approaches to passive immunization

Antigen-Specific Antibody Glycosylation Is Regulated via Vaccination

Antibody effector functions, such as antibody-dependent cellular cytotoxicity, complement deposition, and antibody-dependent phagocytosis, play a critical role in immunity against multiple pathogens, particularly in the absence of neutralizing activity. Two modifications to the IgG constant domain (Fc domain) regulate antibody functionality: changes in antibody subclass and changes in a single N-linked glycan located in the CH2 domain of the IgG Fc. Together, these modifications provide a specific set of instructions to the innate immune system to direct the elimination of antibody-bound antigens. While it is clear that subclass selection is actively regulated during the course of natural infection, it is unclear whether antibody glycosylation can be tuned, in a signal-specific or pathogen-specific manner. Here, we show that antibody glycosylation is determined in an antigen- and pathogen-specific manner during HIV infection. Moreover, while dramatic differences exist in bulk IgG glycosylation among individuals in distinct geographical locations, immunization is able to overcome these differences and elicit antigen-specific antibodies with similar antibody glycosylation patterns. Additionally, distinct vaccine regimens induced different antigen-specific IgG glycosylation profiles, suggesting that antibody glycosylation is not only programmable but can be manipulated via the delivery of distinct inflammatory signals during B cell priming. These data strongly suggest that the immune system naturally drives antibody glycosylation in an antigen-specific manner and highlights a promising means by which next-generation therapeutics and vaccines can harness the antiviral activity of the innate immune system via directed alterations in antibody glycosylation in vivo

Correction: Antigen-Specific Antibody Glycosylation Is Regulated via Vaccination.

[This corrects the article DOI: 10.1371/journal.ppat.1005456.]

Fc Glycan-Mediated Regulation of Placental Antibody Transfer.

Despite the worldwide success of vaccination, newborns remain vulnerable to infections. While neonatal vaccination has been hampered by maternal antibody-mediated dampening of immune responses, enhanced regulatory and tolerogenic mechanisms, and immune system immaturity, maternal pre-natal immunization aims to boost neonatal immunity via antibody transfer to the fetus. However, emerging data suggest that antibodies are not transferred equally across the placenta. To understand this, we used systems serology to define Fc features associated with antibody transfer. The Fc-profile of neonatal and maternal antibodies differed, skewed toward natural killer (NK) cell-activating antibodies. This selective transfer was linked to digalactosylated Fc-glycans that selectively bind FcRn and FCGR3A, resulting in transfer of antibodies able to efficiently leverage innate immune cells present at birth. Given emerging data that vaccination may direct antibody glycosylation, our study provides insights for the development of next-generation maternal vaccines designed to elicit antibodies that will most effectively aid neonates.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Geographic location affects bulk IgG glycosylation.

<p>Bulk IgG glycosylation was assessed in subjects from three regions: Unite States (blue, n = 43), Kenya and Rwanda (maroon, n = 69), and South Africa (yellow, n = 47). (A) Bulk antibody Fc glycosylation in vaccine recipients from each of the three regions was measured via capillary electrophoresis, and the mean proportion of total galactosylated, sialylated, fucosylated, and bisected structures was compared using Kruskal-Wallis one-way ANOVA (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001). (B) Multivariate comparison of antibody Fc glycosylation among the three geographic sites was performed using PCA. The score plot (left panel) depicts the principal component analysis of samples collected in the three regions (each dot represents a vaccinee, and colors are as described above), and the loadings plot of the PCA (right panel) shows the contribution of particular glycan structures to driving the observed separation, where longer arrows signify a greater contribution to separating glycan profiles. This PCA describes 55% of the total variance among these samples.</p

Vaccine-elicited antibody glycosylation profiles are distinct from bulk antibody glycosylation.

<p>Viral vector–induced gp120-specific and influenza specific antibodies were isolated from vaccinees, and the attached glycans were analyzed by capillary electrophoresis. (A) Multivariate PCA was used to compare bulk antibody glycoprofiles (blue, n = 32) and vaccine-elicited antigen-specific antibody glycoprofiles (maroon, n = 20), and both the scores plot (left) and loadings plot (right) are shown. This analysis describes 69% of the variation. (B) The mean proportions of bulk and vaccine-elicited antibody glycan were compared using students two-tailed paired t tests (n = 13 for bulk, n = 20 for gp120 (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001) (C) The mean proportions of vaccine-elicited antibody glycan structures were compared across vaccine groups using Kruskal-Wallis ANOVA (n = 9 for United States, n = 6 for Kenya/Rwanda, n = 4 for South Africa). No statistically significant differences were found. (D) The mean proportions of influenza-specific antibody glycans at baseline (magenta), post-first (green), and post-boost (purple) vaccine timepoints were compared using non-parametric two-way ANOVA (n = 18 for United States, n = 11 for Kenya/Rwanda, n = 5 for South Africa). No significant differences were found between the three timepoints for either antigen or glycan type.</p

Different vaccines induce distinct vaccine-elicited antibody glycosylation profiles.

<p>(A) The antigen-specific IgG glycans from the B003/IPCAVD-004/HVTN 091 (blue) and VAX003 (maroon) studies were compared using PCA. This analysis described 56% of the variation. (B) The mean gp120-specific glycan profiles induced by the B003/IPCAVD-004/HVTN 091 (n = 19) and VAX003 (n = 17) trials were compared using the Mann-Whitney <i>U</i> test (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001).</p