A major obstacle to the development of dengue virus (DENV) vaccines has been the need to immunise concurrently against each of the four DENV serotypes in order to avoid sensitising recipients to developing severe DENV infections. A problem already encountered with live attenuated tetravalent DENV vaccines has been the difficulty in eliciting adequate immune responses against all four DENV serotypes in human hosts. This could have been due to variations in the antigenicity and/or the replication rates of the four DENV serotypes. Non-replicating DNA vaccines avoid the issue of different replication rates. Currently, only DENV-1 and DENV-2 DNA vaccines have been evaluated. In this study, a number of DNA vaccines for each of the four DENV serotypes were developed and their immunogenicity was evaluated in outbred mice. These vaccines included DNA vaccines encoding the DENV prM-E protein genes derived from the four DENV serotypes (pVAX-DEN1, -DEN2, -DEN3 and -DEN4), and DNA vaccines encoding DENV prM and hybrid-E protein genes derived from multiple DENV serotypes. The hybrid-E protein genes were constructed by substituting either domains I and II, domain III, and/or the stem-anchor region from the E protein of one DENV serotype with the corresponding region from another DENV serotype. A number of superior DNA vaccines against each of the four DENV serotypes were identified based on their ability to elicit high titres (≥40, FFURNT50) of neutralising antibodies against the corresponding DENV in mice. The superior DNA vaccines against DENV-1 were pVAX-DEN1, pVAX-C2M2E211, pVAX-C2M2E122 and pVAX-C2M1E122. The superior DNA vaccine against DENV-2 was pVAX-C2M1E122 and the superior DNA vaccines against DENV-3 were pVAX-DEN3 and pVAX-C2M3E344. The superior DNA vaccines against DENV-4 were pVAX-C2M3E344, pVAX-C2M4E434 and pVAX-C2M4E433. Each of these DNA vaccines could provide effective protection against infection by the corresponding DENV serotypes. This is the first study to describe the development of DNA vaccines against DENV-3 and DENV-4. However, mice immunised with a tetravalent DENV DNA vaccine, composed of a DNA vaccine encoding the prM-E protein genes from each of the four DENV serotypes (pVAX-DEN1-4), elicited high titres of neutralising antibodies against DENV-1 and DENV-3 only. Nevertheless, the results from this study suggested that a tetravalent DENV DNA vaccine, composed of pVAX-DEN1, pVAX-C2M1E122, pVAX-DEN3 and pVAX-C2M4E434, may provide effective concurrent protection against infection by each of the four DENV serotypes. In addition, mice immunised with pVAX-C2M1E122, which encoded a hybrid-E protein gene derived from DENV-1 and DENV-2, elicited high titres of anti-DENV-1 and anti-DENV-2 neutralising antibodies, and mice immunised with pVAX-C2M3E344, which encoded a hybrid-E protein gene derived from DENV-3 and DENV-4, elicited high titres of anti-DENV-3 and anti-DENV-4 neutralising antibodies. This result suggested that the co-immunisation of these two hybrid-E DNA vaccines also may provide effective concurrent protection against infection by each of the four DENV serotypes. Extracellular E proteins, believed to be in the form of recombinant subviral particles (RSPs), were recovered from the tissue culture supernatant of all DNA vaccine-transfected mammalian cells by ultracentrifugation, except for cells transfected with the pVAX-C2M2E122 hybrid-E DNA vaccine. Western blotting with the monoclonal antibody 4G2 (flavivirus cross-reactive) demonstrated that the extracellular E proteins expressed by the DNA vaccines were synthesized and cleaved in a manner similar to that of native DENV E proteins. In addition, mammalian cells transfected with pVAX-DEN1, pVAX-DEN2 or pVAX-DEN3 secreted higher amounts of extracellular E proteins than cells transfected with pVAX-DEN4. The amount of extracellular E protein secreted by pVAX-DEN4-transfected cells increased when the c-region of the prM/E signal peptidase cleavage site was made more polar. In contrast, decreasing the polarity of the c-region of the C/prM signal peptidase cleavage site of pVAX-DEN4 resulted in no detectable extracellular E proteins from pVAX-DEN4-transfected cells. This result suggested that the amount of extracellular E proteins secreted by cells transfected with DNA expressing the DENV prM-E protein genes may be dependent of the efficiency of C/prM and prM/E protein cleavages by host-derived signal peptidases. Mice immunised with the mutated pVAX-DEN4, which was capable of expressing large amounts of extracellular E proteins in vitro, produced significantly higher concentrations of Th1-type anti-DENV-4 antibodies than mice immunised with the unmodified pVAX-DEN4, but failed to produce detectable levels of anti-DENV-4 neutralising antibodies. In contrast, increasing the ratio of CpG-S to CpG-N motifs in the pVAX-DEN2 DNA vaccine by incorporating either an additional CpG-S motif, or an antibiotic resistance gene with a high ratio of CpG-S to CpG-N motifs, resulted in a significant increase in both the concentration of Th1-type anti-DENV-2 antibodies and the titres of anti-DENV-2 neutralising antibodies in immunised mice. This result suggested that increasing the amount of CpG-S motifs in DENV DNA vaccines may present an simple and effective approach to increasing the immunogenicity of the DENV DNA vaccines.

Dengue virus (DENV) is the causative agent of dengue fever (DF), the most prevalent arthropod-borne viral disease in the world and therefore is considered an emerging global health threat. The four DENV serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) that infect humans are distinguished from one another by unique antigenic determinants (epitopes) on the DENV envelope (E) protein. The E protein is the primary antigenic site of the DENV and is responsible for inducing neutralising antibody (Ab) and cell mediated immune response in DENV infected hosts. The DENV E protein also mediates attachment of virions to host cell receptors and entry of virions into host cells by membrane fusion. The study of epitopes on DENV E protein is necessary for understanding viral function and for the design of unique polyvalent vaccines capable of inducing a neutralising antibody response against each DENV serotype. Reverse genetics using infectious cDNA clones has enabled the construction of functional intertypic DENV, where the E protein of one DENV serotype is put in the genetic background of a different DENV serotype. In addition, observations from our laboratory indicate that chimeric E proteins, consisting of E protein structural domains from different DENV serotypes can fold into functional proteins. This suggests that there is potential to engineer viruses with intertypic DENV E proteins as potential DENV vaccine candidates, which is the long term goal of studies within our research group. However, if a chimeric E protein was to be constructed containing epitopes involved in antibody mediated neutralisation of each DENV serotype, then knowledge of the location of these epitopes on the E protein of each DENV serotype would be essential. Prior to this study, monoclonal antibodies (MAbs) had been used to identify epitopes involved in antibody mediated neutralisation on the E protein of all DENV serotypes, except DENV-4. The primary objective of this study was to identify epitopes on the DENV-4 E protein involved in neutralisation by antibodies. In order to achieve this objective, a panel of 14 MAbs was generated against DENV-4 in BALB/c mice and characterised using various serological and functional assays. The identification of DENV-4 specific neutralising MAbs in the panel was essential for subsequent experiments aimed at determining antigenic domains, structural domains or specific epitopes (peptides or amino acids) involved in the neutralisation of DENV-4. The majority of MAbs (11/14) generated against DENV-4 recognised the E protein. The remaining three MAbs reacted with the non-structural (NS) 1 protein. The majority of MAbs against the E protein were DENV or Flavivirus group reactive, but four MAbs were DENV-4 specific. All MAbs against the E protein recognised conformationally dependent epitopes and were able to capture DENV-4 in an enzyme linked immuno-adsorbent assay (ELISA). Eighty percent (9/11) of the anti-E MAbs produced for this study neutralised infection of cells by DENV-4 in vitro. Three of the neutralising MAbs (F1G2, 18F5 and 13H8) were DENV-4 specific and also demonstrated the strongest neutralisation activity of the panel, reducing DENV-4 infectivity by 100-1000 fold. The amount of virus neutralised by the MAbs was not related to the avidity of the MAbs. The DENV-4 specific MAbs F1G2, 18F5 and 13H8 were used to identify epitopes involved in neutralisation of DENV-4. The MAbs that effectively captured DENV-4 were used in competitive binding assays (CBAs) to determine spatial relationships between epitopes and therefore define antigenic domains on the DENV-4 E protein. The CBAs indicated that the epitopes recognised by the panel of MAbs segregated into two distinct domains (D4E1 and D4E2) and both contained epitopes involved in neutralisation. CBAs incorporating human serum from DENV-4 infected patients suggested that the MAbs recognised the same, or spatially related, epitopes in domain D4E2 as antibodies from humans who had experienced natural dengue infections, indicating the clinical relevance of such epitopes for the development of DENV vaccines. The reactivity of the capture MAbs with low pH treated DENV-4 was also evaluated in an attempt to identify epitopes that might be more accessible during low pH-mediated virus fusion. Only one of the MAbs (13H8) recognised an acid resistant epitope. Initial attempts to identify epitopes on the DENV-4 E protein involved in neutralisation followed the traditional epitope mapping approach of selecting subpopulations of DENV-4 which escaped neutralisation by MAbs. These attempts were unsuccessful so a variety of strategies for mapping epitopes were used including DENV-4 variant analysis and site directed mutagenesis of the DENV-4 E protein, MAb screening of chimeric DENV-3/4 E proteins and MAb screening of a bacterial peptide display library. DENV-4 variants including DENV-4 isolates from different geographical locations or chemically mutagenised DENV-4 were screened with neutralising MAbs to identify neutralisation escape mutant (n.e.m.) viruses. Site directed mutagenesis of the DENV-4 E protein confirmed whether amino acid changes identified in DENV-4 n.e.m.s were essential for the binding of neutralising MAbs to an epitope. The MAb screening of DENV-4 variants identified n.e.m.s with amino acid changes at residues E95, E96, E156, E157, E203, E329 and E402 of the DENV-4 E protein. Site directed mutagenesis of the DENV-4 E protein identified two epitopes recognised by the DENV-4 specific neutralising MAbs F1G2 and 18F5 at specific amino acid residues within domains II and III of the DENV-4 E protein. No specific epitopes were identified for the MAb 13H8; however this MAb did recognise domain I and II of the DENV-4 E protein, when screened against DENV-3/4 chimeric DENV E proteins. The first epitope, which was recognised by the MAb F1G2, contained residue E95 which was located in domain II of the DENV-4 E protein. The aspartate (Asp) to alanine (Ala) change at E95 prevented the binding of F1G2 to the DENV-4 E protein. The binding of F1G2 to the E95 residue was confirmed using the pFlitrX bacterial peptide display library, which demonstrated binding of F1G2 to a peptide homologous with residues E99-E104. No peptides recognised by 13H8 and 18F5 were identified by this method. The MAb F1G2 also bound to the domain III region (E300-E495) of the DENV-4 E protein when screened against DENV-3/4 chimeric DENV E proteins. This implied that F1G2 may be recognising a discontinuous epitope consisting of domains II and III. The second epitope, which was recognised by MAb 18F5, contained residue E329 which was located in domain III of the DENV-4 E protein. The alanine (Ala) to threonine (Thr) change at E329 prevented the binding of 18F5 to the DENV-4 E protein. MAb 18F5 also bound to the domain III region (E300-E495) of the DENV-4 E protein when screened against DENV-3/4 chimeric E proteins, thus confirming the E329 epitope. The potential mechanisms by which the DENV-4 specific MAbs neutralise virus infection were evaluated by the virus overlay protein binding assay (VOPBA). The binding of MAb 18F5 to a domain III (E329) epitope of the DENV-4 E protein and the binding of MAb F1G2 to domain II (E95, E99-E104) and domain III epitopes (chimeric E protein) of the DENV-4 E protein, prevented the attachment of DENV-4 to a 40 kDa C6/36 cell protein. In contrast the binding of MAb 13H8 to domains I and II of the DENV-4 E protein did not prevent attachment of DENV-4 to the same protein. This was preliminary evidence that the binding of domain III epitopes by the MAbs F1G2 and 18F5 may be important in preventing virus attachment. The binding of MAb 13H8 to domains I and II, and the ability of this MAb to recognise DENV-4 treated at low pH, suggested that MAb 13H8 may block epitopes exposed at low pH that are required for low pH mediated virus fusion to host cell membranes. Overall, the different methods used in this study identified epitopes involved in the neutralisation of DENV-4. The distribution of epitopes involved in neutralisation throughout the DENV-4 E protein were similar to the distribution of epitopes involved in neutralisation on the DENV-1, 2 and 3 E proteins. This suggested that it might be possible to elicit neutralising antibodies against multiple DENV serotypes using chimeric E-proteins derived from two or more DENV serotypes and therefore, facilitate the design of novel tetravalent DENV vaccines.

Since 2012-2013 influenza season, World Health Organization (who) recommends the formulation of tetravalent vaccines. Globally, many countries already use tetravalent vaccines in their national immunization programs, while in Latin America only a small number. Two Influenza b lineages co-circulate, their epidemiological behavior is unpredictable. On average they represent 22.6% of influenza cases and more than 50% in predominant seasons. The lack of concordance between recommended and circulating strains was 25 and 32% in the 2010-2017 and 2000-2013 seasons, respectively. There are no clinical differences between influenza A and B. It occurs more frequently from five to 19 years of age. Influenza b has a higher proportion of attributable deaths than influenza a (1.1 vs. 0.4%), or 2.65 (95% ci 1.18-5.94). A greater number of hospitalizations when the strains mismatch (46.3 vs. 28.5%; p <.0001). Different evaluations have demonstrated its cost effectiveness. The compilation of this information supports the use of quadrivalent vaccines in Latin American countries. / Revisión por pares

A modelagem epidemiológica é uma importante ferramenta que auxilia os órgãos de saúde no controle de doenças infecciosas, pois permitem analisar e comparar diversas estratégias que facilitam a tomada de decisões e definições de protocolos. A dengue é atualmente a doença viral humana com maior número de casos. Possui índice de mortalidade baixo, entretanto, é endêmica em mais de 100 países e 40% da população mundial está em risco de contrair a infecção. Através dos dados de notificação de dengue no Brasil, evidenciamos que os surtos são sazonais, que há alternância de sorotipos ao longo dos anos e mostramos que a doença é diferente em cada localização, e que somente com uma normalização adequada é possível sugerir um agrupamento coerente de municípios. Neste trabalho, as informações obtidas a partir dos dados são usadas para a estruturação dos modelos matemáticos e para a estimação de parâmetros que validam estes modelos. Comparamos a dinâmica de transmissão de dengue do modelo com um sorotipo, com modelos que permitem a interação de dois, três e quatro sorotipos simultaneamente, além da possibilidade de até quatro infecções sequenciais. Os modelos com múltiplos sorotipos são expandidos do modelo básico que categoriza hospedeiros dentro de uma população como suscetíveis (S), infectados (I) e recuperados (R) e acoplado à dinâmica dos vetores suscetíveis (V) e infectados (Vi). Nossos modelos incluem: um período de imunidade cruzada de forma que o indivíduo adquire imunidade permanente para o sorotipo que já foi infectado e imunidade temporária para os demais; uma forçante de sazonalidade na taxa de nascimento dos vetores; uma assimetria com taxas de transmissão diferentes para cada sorotipo; e o compartimento dos vacinados, com uma vacina tetravalente que confere diferentes imunidades para cada sorotipo. Os resultados mostram que para a reprodução de surtos anuais é necessário a inclusão da forçante de sazonalidade na taxa de nascimento dos vetores, e que o modelo com quatro sorotipos é o que melhor reproduz os dados de incidência de dengue, sendo o mais adequado para analisar estratégias de vacinação com uma vacina tetravalente. Comparamos duas estratégias de vacinação: vacinação aleatória na população e vacinação direcionada para faixas etárias. Neste caso, os resultados demonstram a superioridade da estratégia direcionada e que as escolhas das faixas etárias devem ser definidas por município e não por um protocolo nacional. / Epidemiological modelling is an important tool that assists the health agencies in the control of infectious diseases, since it allows analysing and to compare several strategies that facilitate decision-making and protocol definitions. Dengue is currently the most important vector-borne disease. The mortality rate of dengue is low, however, it is endemic in more than 100 countries and about 40% of the world\'s population is at risk of contracting the infection. Through the dengue notification data in Brazil, we emphasize that the outbreaks are seasonal, there is serotypes alternation over the years and we show that the disease is different in each locality, and that only with a suitable standardization it is possible to propose an appropriate grouping of municipalities. In this work, we use the data information to formulate the mathematical models and for the parameter\'s estimation in order to validate these models. We compare the dynamics of dengue of the one serotype model with the models that allow interaction of two, three and four serotypes simultaneously, including the possibility of at most four sequential infections.The multi-strain models are expanded from the basic model which categorizes the host population as susceptible (S), infected (I), and recovered (R) and coupled with the dynamics of the susceptible (V) and infected (Vi) vectors. Our models include: a period of cross-immunity which means permanent immunity to the serotype of the infection and temporary immunity to the other serotypes; a seasonal forcing in the mosquitoes birth rate; different transmissions rates, so that the models are asymmetric; and the compartment of vaccinated individuals with a tetravalent vaccine which confers different immunities for each serotype. The results show that to reproduce yearly outbreaks it is necessary to include the seasonal forcing in the birth rate of the vectors, and that the four serotypes model is the one that best reproduces the dengue incidence data, being the most suitable model to analyse vaccination strategies with a tetravalent vaccine. We compare two vaccination strategies: random vaccination and vaccination targeted at age groups. In this case, the results demonstrate the superiority of the targeted strategy and that the choices of the age groups should be defined by municipality and not by a national protocol.

A modelagem epidemiológica é uma importante ferramenta que auxilia os órgãos de saúde no controle de doenças infecciosas, pois permitem analisar e comparar diversas estratégias que facilitam a tomada de decisões e definições de protocolos. A dengue é atualmente a doença viral humana com maior número de casos. Possui índice de mortalidade baixo, entretanto, é endêmica em mais de 100 países e 40% da população mundial está em risco de contrair a infecção. Através dos dados de notificação de dengue no Brasil, evidenciamos que os surtos são sazonais, que há alternância de sorotipos ao longo dos anos e mostramos que a doença é diferente em cada localização, e que somente com uma normalização adequada é possível sugerir um agrupamento coerente de municípios. Neste trabalho, as informações obtidas a partir dos dados são usadas para a estruturação dos modelos matemáticos e para a estimação de parâmetros que validam estes modelos. Comparamos a dinâmica de transmissão de dengue do modelo com um sorotipo, com modelos que permitem a interação de dois, três e quatro sorotipos simultaneamente, além da possibilidade de até quatro infecções sequenciais. Os modelos com múltiplos sorotipos são expandidos do modelo básico que categoriza hospedeiros dentro de uma população como suscetíveis (S), infectados (I) e recuperados (R) e acoplado à dinâmica dos vetores suscetíveis (V) e infectados (Vi). Nossos modelos incluem: um período de imunidade cruzada de forma que o indivíduo adquire imunidade permanente para o sorotipo que já foi infectado e imunidade temporária para os demais; uma forçante de sazonalidade na taxa de nascimento dos vetores; uma assimetria com taxas de transmissão diferentes para cada sorotipo; e o compartimento dos vacinados, com uma vacina tetravalente que confere diferentes imunidades para cada sorotipo. Os resultados mostram que para a reprodução de surtos anuais é necessário a inclusão da forçante de sazonalidade na taxa de nascimento dos vetores, e que o modelo com quatro sorotipos é o que melhor reproduz os dados de incidência de dengue, sendo o mais adequado para analisar estratégias de vacinação com uma vacina tetravalente. Comparamos duas estratégias de vacinação: vacinação aleatória na população e vacinação direcionada para faixas etárias. Neste caso, os resultados demonstram a superioridade da estratégia direcionada e que as escolhas das faixas etárias devem ser definidas por município e não por um protocolo nacional. / Epidemiological modelling is an important tool that assists the health agencies in the control of infectious diseases, since it allows analysing and to compare several strategies that facilitate decision-making and protocol definitions. Dengue is currently the most important vector-borne disease. The mortality rate of dengue is low, however, it is endemic in more than 100 countries and about 40% of the world\'s population is at risk of contracting the infection. Through the dengue notification data in Brazil, we emphasize that the outbreaks are seasonal, there is serotypes alternation over the years and we show that the disease is different in each locality, and that only with a suitable standardization it is possible to propose an appropriate grouping of municipalities. In this work, we use the data information to formulate the mathematical models and for the parameter\'s estimation in order to validate these models. We compare the dynamics of dengue of the one serotype model with the models that allow interaction of two, three and four serotypes simultaneously, including the possibility of at most four sequential infections.The multi-strain models are expanded from the basic model which categorizes the host population as susceptible (S), infected (I), and recovered (R) and coupled with the dynamics of the susceptible (V) and infected (Vi) vectors. Our models include: a period of cross-immunity which means permanent immunity to the serotype of the infection and temporary immunity to the other serotypes; a seasonal forcing in the mosquitoes birth rate; different transmissions rates, so that the models are asymmetric; and the compartment of vaccinated individuals with a tetravalent vaccine which confers different immunities for each serotype. The results show that to reproduce yearly outbreaks it is necessary to include the seasonal forcing in the birth rate of the vectors, and that the four serotypes model is the one that best reproduces the dengue incidence data, being the most suitable model to analyse vaccination strategies with a tetravalent vaccine. We compare two vaccination strategies: random vaccination and vaccination targeted at age groups. In this case, the results demonstrate the superiority of the targeted strategy and that the choices of the age groups should be defined by municipality and not by a national protocol.

Synthese und Charakterisierung (SC-XRD, P-XRD, IR, NMR, QCC) metall-organischer Komplexe tetravalenter Actinide Th, U, Np, Pu mit Liganden Salen und Derivat Salpn im Festkörper und in Lösung.

Pas de résumé en français / The goal of this research was the synthesis and spectroscopic, structural andphysical-chemical characterization of cyclic 6- and 7-membered hydroxamicacids, a tetrahydroxamic calix[4]arene-based tetrapodal receptor, and their metalcomplexes with trivalent and tetravalent metal cations. They were characterizedby several techniques such as 1H and 13C NMR, IR, and mass spectroscopies,single crystal X-ray analysis, and potentiometry. Cyclic hydroxamic acids arefound in a few mix siderophores but their coordination properties were stillunknown. The structural features of metal complexes formed with Fe(III),Ga(III), Ce(IV), Zr(IV), Hf(IV), U(IV) and U(VI) have been investigated both inthe solid state and in solution. The synthesis and complexation studies of anoriginal calix[4]arene-based tetrapodal receptor is described. Reactionparameters for all key steps in the synthetic route have been optimized. Thesingle X-ray crystal analysis of benzyl-protected receptor was obtained.Complexation studies with zirconium(IV) and hafnium(IV) evidenced theformation of two metal two ligand complexes rather than 1:1 species, whichwere shown to interact in solution with a third alkali cation

To study the functions of glycoconjugates in biological systems reliable and efficient protocols for glycoconjugate synthesis are needed. To reach this goal we have developed methods for solid-phase synthesis of glycoconjugates that can be monitored with gel-phase 19F spectroscopy using fluorinated linkers, building blocks, and protecting groups. We have developed a new fluorine containing linker suitable for solid-phase synthesis of glycoconjugates. The linker was more acid-labile than similar linkers in order to enable cleavage under mild conditions of the target compound from the linker resin.  A carbamate-based strategy has been applied to attach a spacer carrying an amino group to a fluorinated Wang linker for synthesis of amino-functionalized glycoconjugates using thioglycoside donors with fluorinated protective groups. Cleavage from the solid support was performed with trifluoroacetic acid and subsequent protecting group removal gave the target compound. The terminal amine was conjugated with didecyl squarate and this derivative can be attached to various proteins and solid surfaces carrying primary or secondary amines. To evaluate this methodology we have immobilized glycoconjugates in amino-functionalized microtiter plates and successfully probed them with lectin. In addition, a novel fluorine containing protecting group has been designed, synthesized and evaluated. The protecting group was used for protection of the unreactive 4-OH in a galactose building block that was applied in the synthesis of 6-aminohexyl galabioside and was removed with TBAF in THF. Adenovirus serotype 8 (Ad8), Ad19, and Ad37 cause the severe ocular infection, epidemic keratoconjunctivities (EKC). During infection, the adenoviruses interact with sialic acid containing glycoconjugates on the epithelial cells via fiber structures extending from the viral particles. The virus particle most likely binds to the host cell in a multivalent way by simultaneously using multiple fiber proteins and binding sites. Multivalent sialic acid containing conjugates could efficiently inhibit Ad37 cell attachment and subsequent infection of human corneal epithelial (HCE) cells. Three compact tri- and tetravalent sialic acid conjugates were prepared and evaluated as inhibitors of adenoviral host cell attachment and subsequent infection and all conjugates were potent as anti-adenoviral agents. The conjugates can readily be synthesized from accessible starting materials. A crystal structure of the Ad37 fiber knob protein and the trivalent sialic acid conjugate showed that the three binding sites were all occupied by one sialic acid residue each.