Application and comparison of lyophilisation protocols to enhance stable long-term storage of filovirus pseudotypes for use in antibody neutralisation tests.

AIMS: Filoviruses encompass highly pathogenic viruses placing significant public health burden on countries affected. Efforts for improved diagnostics and surveillance are needed. The requirement for high-containment can be circumvented by using pseudotype viruses (PV), which can be handled safely, in tropism, drug screening, vaccine evaluation, and serosurveillance studies. We assessed the stability and functionality after long-term storage of lyophilised filovirus pseudotypes for use in neutralisation assays. METHODS AND RESULTS: We generated a panel of filovirus lentiviral pseudotypes followed by lyophilisation and storage in different conditions. Next, we reconstituted and tested PVs in infection experiments and pseudotype neutralisation assays where possible. Lyophilised Ebola and Marburg PVs retained production titres for at least two years when stored at +4˚C or less. Lyophilised Ebola PVs performed similarly to non-lyophilised PVs in neutralisation assays after reconstitution. When stored at high temperatures (+37˚C), lyophilised PVs did not retain titres after 1-month storage, however, when lyophilised using pilot-scale facilities EBOV PVs retained titres and performed as standard in neutralisation assays after on 1-month storage at 37˚C. CONCLUSIONS: Filovirus PVs are amenable to lyophilisation and can be stored for at least 2 years in a household fridge to be used in antibody assays. Lyophilisation performed in the right conditions would allow transportation at room temperature, even in warmer climates.

Intranasal administration of a live-attenuated recombinant newcastle disease virus expressing the SARS-CoV-2 Spike protein induces high neutralizing antibody levels and protects from experimental challenge infection in hamsters.

The emergence of SARS-CoV-2 in December 2019 resulted in the COVID-19 pandemic. Recurring disease outbreaks repeatedly overloaded the public health sector and severely affected the global economy. We developed a candidate COVID-19 vaccine based on a recombinant Newcastle disease virus (NDV) vaccine vector, encoding a pre-fusion stabilized full-length Spike protein obtained from the original SARS-CoV-2 Wuhan isolate. Vaccination of hamsters by intra-muscular injection or intra-nasal instillation induced high neutralizing antibody responses. Intranasal challenge infection with SARS-CoV-2 strain Lelystad demonstrated that both vaccination routes provided partial protection in the upper respiratory tract, and almost complete protection in the lower respiratory tract, as measured by suppressed viral loads and absence of histological lung lesions. Activity wheel measurements demonstrated that animals vaccinated by intranasal inoculation rapidly recovered to normal activity. NDV constructs encoding the spike of SARS-CoV-2 may be attractive candidates for development of intra-nasal COVID-19 booster vaccines.

Activation of Human Monocytes by Colloidal Aluminum Salts.

Subunit vaccines often contain colloidal aluminum salt-based adjuvants to activate the innate immune system. These aluminum salts consist of micrometer-sized aggregates. It is well-known that particle size affects the adjuvant effect of particulate adjuvants. In this study, the activation of human monocytes by hexagonal-shaped gibbsite (ø = 210 ± 40 nm) and rod-shaped boehmite (ø = 83 ± 827 nm) was compared with classical aluminum oxyhydroxide adjuvant (alum). To this end, human primary monocytes were cultured in the presence of alum, gibbsite, or boehmite. The transcriptome and proteome of the monocytes were investigated by using quantitative polymerase chain reaction and mass spectrometry. Human monocytic THP-1 cells were used to investigate the effect of the particles on cellular maturation, differentiation, activation, and cytokine secretion, as measured by flow cytometry and enzyme-linked immunosorbent assay. Each particle type resulted in a specific gene expression profile. IL-1ß and IL-6 secretion was significantly upregulated by boehmite and alum. Of the 7 surface markers investigated, only CD80 was significantly upregulated by alum and none by gibbsite or boehmite. Gibbsite hardly activated the monocytes. Boehmite activated human primary monocytes equally to alum, but induced a much milder stress-related response. Therefore, boehmite was identified as a promising adjuvant candidate.

Skin delivery of trivalent Sabin inactivated poliovirus vaccine using dissolvable microneedle patches induces neutralizing antibodies.

The cessation of the oral poliovirus vaccine (OPV) and the inclusion of inactivated poliovirus (IPV) into all routine immunization programmes, strengthens the need for new IPV options. Several novel delivery technologies are being assessed that permit simple yet efficacious and potentially dose-sparing administration of IPV. Current disadvantages of conventional liquid IPV include the dependence on cold chain and the need for injection, resulting in high costs, production of hazardous sharps waste and requiring sufficiently trained personnel. In the current study, a dissolvable microneedle (DMN) patch for skin administration that incorporates trivalent inactivated Sabin poliovirus vaccine (sIPV) was developed. Microneedles were physically stable in the ambient environment for at least 30 min and efficiently penetrated skin. Polio-specific IgG antibodies that were able to neutralize the virus were induced in rats upon administration using trivalent sIPV-containing microneedle patches. These sIPV-patch-induced neutralizing antibody responses were comparable to higher vaccine doses delivered intramuscularly for type 1 and type 3 poliovirus serotypes. Moreover, applying the patches to the flank elicited a significantly higher antibody response compared to their administration to the ear. This study progresses the development of a skin patch-based technology that would simplify vaccine administration of Sabin IPV and thereby overcome logistic issues currently constraining poliovirus eradication campaigns.

Stabilised aluminium phosphate nanoparticles used as vaccine adjuvant.

Aluminium phosphate is a commonly used adjuvant consisting of heterogeneously sized aggregates up to several micrometers. However, aluminium phosphate nanoparticles may exhibit an improved adjuvant effect. In this study, nanoparticles were made by sonication of commercially available aluminium phosphate adjuvant, resulting in particles with a size (Z-average diameter) between 200-300 nm and a point of zero charge of 4.5. To prevent reaggregation, which occurred within 14 days, a screening of excipients was performed to identify stabilisers effective under physiological conditions (pH 7.4, 290 mOsm). The amino acids threonine, asparagine, and L-alanyl-L-1-aminoethylphosphonic acid (LAPA) stabilised sonicated aluminium phosphate. Particle sizes remained stable between 400-600 nm at 37 °C during 106 days. Contrarily, arginine induced strong reaggregation to a particle size larger than 1000 nm. The stability of aluminium phosphate nanoparticles was strongly affected by the pH. Aggregation mainly occurred below pH 7. The adsorption capacity, a potentially relevant parameter for adjuvants, was slightly reduced in the presence of asparagine, when using a model antigen (lysozyme). LAPA, arginine, threonine and aspartic acid reduced protein adsorption significantly. The adjuvant effect of aluminium phosphate nanoparticles was studied by immunisation of mice with diphtheria toxoid adjuvanted with the aluminium phosphate nanoparticles. The presence of LAPA, threonine, aspartic acid or asparagine did not alter diphtheria toxoid-specific antibody or toxin-neutralising antibody titres. Arginine increased diphtheria toxoid-specific antibody titres but not toxin-neutralising antibody titres. In conclusion, aluminium phosphate nanoparticles were stabilised by particular amino acids and induced an adjuvant effect comparable to that of aluminium phosphate microparticles.

Development of a thermostable spray dried outer membrane vesicle pertussis vaccine for pulmonary immunization.

Worldwide resurgence of whooping cough calls for improved, next-generation pertussis vaccines that induce broad and long-lasting immunity. A mucosal pertussis vaccine based on outer membrane vesicles (omvPV) is a promising candidate. Further, a vaccine that is stable outside the cold chain would be of substantial advantage for worldwide distribution and application. A vaccine formulated as a powder could both stabilize the vaccine as well as make it suitable for pulmonary vaccination. To that end, we developed a spray dried omvPV with improved stability compared to the liquid omvPV formulation. Spray drying did not affect the structural integrity of the omvPV. The antigenicity of Vag8, a major antigen in omvPV was diminished slightly and an altered tryptophan fluorescence indicated some changes in protein structure. However, when administered via the pulmonary route in mice after reconstitution, spray dried omvPV showed comparable immune responses and protection against challenge with live B. pertussis as liquid omvPV. Mucosal IgA and Th17 responses were established in addition to broad systemic IgG and Th1/Th17 responses, indicating the induction of an effective immunity profile. Overall, a spray dried omvPV was developed that maintained effective immunogenic properties and has an improved storage stability.

The effect of formulation on spray dried Sabin inactivated polio vaccine.

The objective of this study was to develop a stable spray dried formulation, containing the three serotypes of Sabin inactivated polio vaccine (sIPV), aiming for minimal loss of native conformation (D-antigen) during drying and subsequent storage. The influence of atomization and drying stress during spray drying on trivalent sIPV was investigated. This was followed by excipient screening, in which monovalent sIPV was formulated and spray dried. Excipient combinations and concentrations were tailored to maximize both the antigen recovery of respective sIPV serotypes after spray drying and storage (T = 40 °C and t = 7 days). Furthermore, a fractional factorial design was developed around the most promising formulations to elucidate the contribution of each excipient in stabilizing D-antigen during drying. Serotype 1 and 2 could be dried with 98% and 97% recovery, respectively. When subsequently stored at 40 °C for 7 days, the D-antigenicity of serotype 1 was fully retained. For serotype 2 the D-antigenicity dropped to 71%. Serotype 3 was more challenging to stabilize and a recovery of 56% was attained after drying, followed by a further loss of 37% after storage at 40 °C for 7 days. Further studies using a design of experiments approach demonstrated that trehalose/monosodium glutamate and maltodextrin/arginine combinations were crucial for stabilizing serotype 1 and 2, respectively. For sIPV serotype 3, the best formulation contained Medium199, glutathione and maltodextrin. For the trivalent vaccine it is therefore probably necessary to spray dry the different serotypes separately and mix the dry powders afterwards to obtain the trivalent vaccine.

Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes.

Influenza peptide antigens coding for conserved T cell epitopes have the capacity to induce cross-protective influenza-specific immunity. Short peptide antigens used as a vaccine, however, often show poor immunogenicity. In this study, we demonstrate that whole-inactivated influenza virus (WIV) acts as an adjuvant for influenza peptide antigens, as shown by the induction of peptide-specific CD8+ T cells in HLA-A2.1 transgenic mice upon vaccination with the influenza-M1-derived GILGFVFTL peptide (GIL), formulated with WIV. By screening various concentrations of GIL and WIV, we found that both components contributed to the GIL-specific T cell response. Whereas co-localization of the peptide antigen and WIV adjuvant was found to be important, neither physical association between peptide and WIV nor fusogenic activity of WIV were relevant for the adjuvant effect of WIV. We furthermore show that WIV may adjuvate T cell responses to a variety of peptides, using pools of either conserved wild-type influenza peptides or chemically altered peptide ligands. This study shows the potential of WIV as an adjuvant for influenza peptides. The simple formulation process and the solid safety record of WIV make this an attractive adjuvant for T cell peptides, and may also be used for non-influenza antigens.

Developments in the formulation and delivery of spray dried vaccines.

Spray drying is a promising method for the stabilization of vaccines, which are usually formulated as liquids. Usually, vaccine stability is improved by spray drying in the presence of a range of excipients. Unlike freeze drying, there is no freezing step involved, thus the damage related to this step is avoided. The edge of spray drying resides in its ability for particles to be engineered to desired requirements, which can be used in various vaccine delivery methods and routes. Although several spray dried vaccines have shown encouraging preclinical results, the number of vaccines that have been tested in clinical trials is limited, indicating a relatively new area of vaccine stabilization and delivery. This article reviews the current status of spray dried vaccine formulations and delivery methods. In particular it discusses the impact of process stresses on vaccine integrity, the application of excipients in spray drying of vaccines, process and formulation optimization strategies based on Design of Experiment approaches as well as opportunities for future application of spray dried vaccine powders for vaccine delivery.

Intranasal and sublingual delivery of inactivated polio vaccine.

Polio is on the brink of eradication. Improved inactivated polio vaccines (IPV) are needed towards complete eradication and for the use in the period thereafter. Vaccination via mucosal surfaces has important potential advantages over intramuscular injection using conventional needle and syringe, the currently used delivery method for IPV. One of them is the ability to induce both serum and mucosal immune responses: the latter may provide protection at the port of virus entry. The current study evaluated the possibilities of polio vaccination via mucosal surfaces using IPV based on attenuated Sabin strains. Mice received three immunizations with trivalent sIPV via intramuscular injection, or via the intranasal or sublingual route. The need of an adjuvant for the mucosal routes was investigated as well, by testing sIPV in combination with the mucosal adjuvant cholera toxin. Both intranasal and sublingual sIPV immunization induced systemic polio-specific serum IgG in mice that were functional as measured by poliovirus neutralization. Intranasal administration of sIPV plus adjuvant induced significant higher systemic poliovirus type 3 neutralizing antibody titers than sIPV delivered via the intramuscular route. Moreover, mucosal sIPV delivery elicited polio-specific IgA titers at different mucosal sites (IgA in saliva, fecal extracts and intestinal tissue) and IgA-producing B-cells in the spleen, where conventional intramuscular vaccination was unable to do so. However, it is likely that a mucosal adjuvant is required for sublingual vaccination. Further research on polio vaccination via sublingual mucosal route should include the search for safe and effective adjuvants, and the development of novel oral dosage forms that improve antigen uptake by oral mucosa, thereby increasing vaccine immunogenicity. This study indicates that both the intranasal and sublingual routes might be valuable approaches for use in routine vaccination or outbreak control in the period after complete OPV cessation and post-polio eradication.

A Design of Experiment approach to predict product and process parameters for a spray dried influenza vaccine.

Spray dried vaccine formulations might be an alternative to traditional lyophilized vaccines. Compared to lyophilization, spray drying is a fast and cheap process extensively used for drying biologicals. The current study provides an approach that utilizes Design of Experiments for spray drying process to stabilize whole inactivated influenza virus (WIV) vaccine. The approach included systematically screening and optimizing the spray drying process variables, determining the desired process parameters and predicting product quality parameters. The process parameters inlet air temperature, nozzle gas flow rate and feed flow rate and their effect on WIV vaccine powder characteristics such as particle size, residual moisture content (RMC) and powder yield were investigated. Vaccine powders with a broad range of physical characteristics (RMC 1.2-4.9%, particle size 2.4-8.5µm and powder yield 42-82%) were obtained. WIV showed no significant loss in antigenicity as revealed by hemagglutination test. Furthermore, descriptive models generated by DoE software could be used to determine and select (set) spray drying process parameter. This was used to generate a dried WIV powder with predefined (predicted) characteristics. Moreover, the spray dried vaccine powders retained their antigenic stability even after storage for 3 months at 60°C. The approach used here enabled the generation of a thermostable, antigenic WIV vaccine powder with desired physical characteristics that could be potentially used for pulmonary administration.

Predicting the influence of liposomal lipid composition on liposome size, zeta potential and liposome-induced dendritic cell maturation using a design of experiments approach.

In this study, the effect of liposomal lipid composition on the physicochemical characteristics and adjuvanticity of liposomes was investigated. Using a design of experiments (DoE) approach, peptide-containing liposomes containing various lipids (EPC, DOPE, DOTAP and DC-Chol) and peptide concentrations were formulated. Liposome size and zeta potential were determined for each formulation. Moreover, the adjuvanticity of the liposomes was assessed in an in vitro dendritic cell (DC) model, by quantifying the expression of DC maturation markers CD40, CD80, CD83 and CD86. The acquired data of these liposome characteristics were successfully fitted with regression models, and response contour plots were generated for each response factor. These models were applied to predict a lipid composition that resulted in a liposome with a target zeta potential. Subsequently, the expression of the DC maturation factors for this lipid composition was predicted and tested in vitro; the acquired maturation responses corresponded well with the predicted ones. These results show that a DoE approach can be used to screen various lipids and lipid compositions, and to predict their impact on liposome size, charge and adjuvanticity. Using such an approach may accelerate the formulation development of liposomal vaccine adjuvants.

Development of cross-protective influenza a vaccines based on cellular responses.

Seasonal influenza vaccines provide protection against matching influenza A virus (IAV) strains mainly through the induction of neutralizing serum IgG antibodies. However, these antibodies fail to confer a protective effect against mismatched IAV. This lack of efficacy against heterologous influenza strains has spurred the vaccine development community to look for other influenza vaccine concepts, which have the ability to elicit cross-protective immune responses. One of the concepts that is currently been worked on is that of influenza vaccines inducing influenza-specific T cell responses. T cells are able to lyse infected host cells, thereby clearing the virus. More interestingly, these T cells can recognize highly conserved epitopes of internal influenza proteins, making cellular responses less vulnerable to antigenic variability. T cells are therefore cross-reactive against many influenza strains, and thus are a promising concept for future influenza vaccines. Despite their potential, there are currently no T cell-based IAV vaccines on the market. Selection of the proper antigen, appropriate vaccine formulation and evaluation of the efficacy of T cell vaccines remains challenging, both in preclinical and clinical settings. In this review, we will discuss the current developments in influenza T cell vaccines, focusing on existing protein-based and novel peptide-based vaccine formulations. Furthermore, we will discuss the feasibility of influenza T cell vaccines and their possible use in the future.

Current and next generation influenza vaccines: Formulation and production strategies.

Vaccination is the most effective method to prevent influenza infection. However, current influenza vaccines have several limitations. Relatively long production times, limited vaccine capacity, moderate efficacy in certain populations and lack of cross-reactivity are important issues that need to be addressed. We give an overview of the current status and novel developments in the landscape of influenza vaccines from an interdisciplinary point of view. The feasibility of novel vaccine concepts not only depends on immunological or clinical outcomes, but also depends on biotechnological aspects, such as formulation and production methods, which are frequently overlooked. Furthermore, the next generation of influenza vaccines is addressed, which hopefully will bring cross-reactive influenza vaccines. These developments indicate that an exciting future lies ahead in the influenza vaccine field.

Influenza T-cell epitope-loaded virosomes adjuvanted with CpG as a potential influenza vaccine.

PURPOSE: Influenza CD8(+) T-cell epitopes are conserved amongst influenza strains and can be recognized by influenza-specific cytotoxic T-cells (CTLs), which can rapidly clear infected cells. An influenza peptide vaccine that elicits these CTLs would therefore be an alternative to current influenza vaccines, which are not cross-reactive. However, peptide antigens are poorly immunogenic due to lack of delivery to antigen presenting cells, and therefore need additional formulation with a suitable delivery system. In this study, the potential of virosomes as a delivery system for an influenza T-cell peptide was investigated. METHODS: The conserved human HLA-A2.1 influenza T-cell epitope M158-66 was formulated with virosomes. The immunogenicity and protective effect of the peptide-loaded virosomes was assessed in HLA-A2 transgenic mice. Delivery properties of the virosomes were studied in mice and in in vitro dendritic cell cultures. RESULTS: Immunization of HLA-A2.1 transgenic C57BL/6 mice with peptide-loaded virosomes in the presence of the adjuvant CpG-ODN 1826 increased the number of peptide-specific CTLs. Vaccination with adjuvanted peptide-loaded virosomes reduced weight loss in mice after heterologous influenza infection. Association with fusion-active virosomes was found to be crucial for antigen uptake by dendritic cells, and subsequent induction of CTLs in mice. CONCLUSIONS: These results show that influenza virosomes loaded with conserved influenza epitopes could be the basis of a novel cross-protective influenza vaccine.

Solid bioneedle-delivered influenza vaccines are highly thermostable and induce both humoral and cellular immune responses.

The potential of bioneedles to deliver influenza vaccines was investigated. Four influenza vaccine formulations were screened to determine the optimal formulation for use with bioneedles. The stability of the formulations after freeze-drying was checked to predict the stability of the influenza vaccines in the bioneedles. Subunit, split, virosomal and whole inactivated influenza (WIV) vaccine were formulated and lyophilized in bioneedles, and subsequently administered to C57BL/6 mice. Humoral and cellular immune responses were assessed after vaccination. The thermostability of lyophilized vaccines was determined after one-month storage at elevated temperatures. Bioneedle influenza vaccines induced HI titers that are comparable to those induced by intramuscular WIV vaccination. Delivery by bioneedles did not alter the type of immune response induced by the influenza vaccines. Stability studies showed that lyophilized influenza vaccines have superior thermostability compared to conventional liquid vaccines, and remained stable after one-month storage at 60°C. Influenza vaccines delivered by bioneedles are a viable alternative to conventional liquid influenza vaccines. WIV was determined to be the most potent vaccine formulation for administration by bioneedles. Lyophilized influenza vaccines in bioneedles are independent of a cold-chain, due to their increased thermostability, which makes distribution and stockpiling easier.

Focused magnetic stem cell targeting to the retina using superparamagnetic iron oxide nanoparticles.

Developing new ways of delivering cells to diseased tissue will be a key factor in translating cell therapeutics research into clinical use. Magnetically targeting cells enables delivery of significant numbers of cells to key areas of specific organs. To demonstrate feasibility in neurological tissue, we targeted cells magnetically to the upper hemisphere of the rodent retina. Rat mesenchymal stem cells (MSCs) were magnetized using superparamagnetic iron oxide nanoparticles (SPIONs). In vitro studies suggested that magnetization with fluidMAG-D was well tolerated, that cells remained viable, and they retained their differentiation capabilities. FluidMAG-D-labeled MSCs were injected intravitreally or via the tail vein of the S334ter-4 transgenic rat model of retinal degeneration with or without placing a gold-plated neodymium disc magnet within the orbit, but outside the eye. Retinal flatmount and cryosection imaging demonstrated that after intravitreal injection cells localized to the inner retina in a tightly confined area corresponding to the position of the orbital magnet. After intravenous injection, similar retinal localization was achieved and remarkably was associated with a tenfold increase in magnetic MSC delivery to the retina. Cryosections demonstrated that cells had migrated into both the inner and outer retina. Magnetic MSC treatment with orbital magnet also resulted in significantly higher retinal concentrations of anti-inflammatory molecules interleukin-10 and hepatocyte growth factor. This suggested that intravenous MSC therapy also resulted in significant therapeutic benefit in the dystrophic retina. With minimal risk of collateral damage, these results suggest that magnetic cell delivery is the best approach for controlled delivery of cells to the outer retina-the focus for disease in age-related macular degeneration and retinitis pigmentosa.

Hyperbranched polyglycerols as trimodal imaging agents: design, biocompatibility, and tumor uptake.

Combining various imaging modalities often leads to complementary information and synergistic advantages. A trimodal long-circulating imaging agent tagged with radioactive, magnetic resonance, and fluorescence markers is able to combine the high sensitivity of SPECT with the high resolution of MRI over hours and days. The fluorescence marker helps to confirm the in vivo imaging information at the microscopic level, in the context of the tumor microenvironment. To make a trimodal long-circulating probe, high-molecular-weight hyperbranched polyglycerols (HPG) were modified with a suitable ligand for (111)In radiolabeling and Gd coordination, and additionally tagged with a fluorescent dye. The resulting radiopharmaceutical and contrast agent was nontoxic and hemocompatible. Measured radioactively, its total tumor uptake increased from 2.6% at 24 h to 7.3% at 72 h, which is twice the increase expected due to tumor growth in this time period. Both in vivo MRI and subsequent histological analyses of the same tumors confirmed maximum HPG accumulation at 3 days post injection. Furthermore, Gd-derivatized HPG has an excellent contrast enhancement on T1-weighted MRI at 10× lower molar concentrations than commercially available Galbumin. HPG derivatized with gadolinium, radioactivity, and fluorescence are thus long-circulating macromolecules with great potential for imaging of healthy and leaky blood vessels using overlapping multimodal approaches and for the passive targeting of tumors.

Conjugation of ovalbumin to trimethyl chitosan improves immunogenicity of the antigen.

Subunit vaccines are generally safer, but often less effective than live attenuated vaccines as they lack the necessary co-stimulatory factors. The formulation of an adjuvant like N-trimethyl chitosan (TMC) with an antigen can overcome its poor immunogenicity. Recent data suggest the importance of incorporating the antigen and the adjuvant into one entity for maximum immunostimulatory effect, e.g. by using (nano)particles. In the present paper we introduce the conjugation of an antigen, ovalbumin (OVA), to TMC as an alternative to nanoparticles for subunit vaccination. OVA was covalently linked to TMC using thiol chemistry (SPDP method). The uptake of the resulting TMC-OVA conjugate by dendritic cells (DC) and its effect on DC maturation was assessed in vitro and its immunogenicity was investigated in mice. We found that with the SPDP method a reducible covalent bond between TMC and OVA could be introduced, without disrupting the protein's antigenicity and structure. Uptake of TMC-OVA conjugate by dendritic cells was similar to the uptake of TMC/OVA nanoparticles, over 5-fold increased compared to a solution of OVA and TMC. Mice immunized with TMC-OVA conjugate produced 1000-fold higher OVA specific IgG titers than mice immunized with either OVA or a physical mixture of TMC and OVA. Moreover, these antibody titers were slightly elevated compared to the titers obtained with TMC/OVA nanoparticles. Conjugation of the antigen to an adjuvant is therefore a viable strategy to increase the immunogenicity of subunit vaccines and may provide an alternative to the use of particles.