Dengue NS1 antibodies are associated with clearance of viral NS1.

BACKGROUND: Dengue vascular permeability syndrome is the primary cause of death in severe dengue infections. The protective versus potentially pathogenic role of dengue NS1 antibodies are not well understood. The main goal of this analysis was to characterize the relationship between free NS1 concentration and NS1 antibody titers in primary and secondary dengue infection in order to better understand the presence and duration of NS1 antibody complexes in clinical dengue infections. METHODS: Hospitalized participants with acute dengue infection were recruited from Northern Colombia between 2018 to 2020. Symptom assessment including dengue signs and symptoms, chart review and blood collection was performed. Primary versus secondary Dengue was assessed serologically. NS1 titers and anti-NS1 antibodies were measured daily. RESULTS: Patients with secondary infection have higher antibody titers than those in primary infection, and we find a negative correlation between anti-NS1 antibody titer and NS1 protein. We demonstrate that in a subset of secondary infection, there are indeed NS1 antibody-antigen complexes at the admission day during the febrile phase that are not detectable by the recovery phase. Furthermore, dengue infection status is associated with higher circulating sialidases. DISCUSSION: The negative correlation between antibody and protein suggests that antibodies may play a role in clearing this viral protein.

DNA Vaccine Delivery and Improved Immunogenicity.

The promise of DNA vaccines is as compelling today as it was more than a decade ago. Ease of manufacture, stability at ambient temperatures without the need for a cold chain and its ability to mimic natural infections and elicit appropriate immune responses makes this vaccine platform extremely attractive. Although, human clinical trials of DNA vaccines have yielded less than optimal results, the approval and licensing of a few veterinary vaccines is testimony to the proof-of-concept and the hope that licensed DNA vaccines for human use may not be too far away. Delivery and targeting of immunologically relevant cells appears to be the major hurdle in maximizing the immunogenicity of DNA vaccines. Several different approaches that are currently pursued in achieving this objective are discussed.

Immunogenicity and Protective Efficacy of Psoralen-Inactivated SARS-CoV-2 Vaccine in Nonhuman Primates.

COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has significantly impacted public health and the economy worldwide. Most of the currently licensed COVID-19 vaccines act by inhibiting the receptor-binding function of the SARS-CoV-2 spike protein. The constant emergence of SARS-CoV-2 variants resulting from mutations in the receptor-binding domain (RBD) leads to vaccine immune evasion and underscores the importance of broadly acting COVID-19 vaccines. Inactivated whole virus vaccines can elicit broader immune responses to multiple epitopes of several antigens and help overcome such immune evasions. We prepared a psoralen-inactivated SARS-CoV-2 vaccine (SARS-CoV-2 PsIV) and evaluated its immunogenicity and efficacy in nonhuman primates (NHPs) when administered with the Advax-CpG adjuvant. We also evaluated the SARS-CoV-2 PsIV as a booster shot in animals vaccinated with a DNA vaccine that can express the full-length spike protein. The Advax-CpG-adjuvanted SARS-CoV-2 PsIV elicited a dose-dependent neutralizing antibody response in the NHPs, as measured using a serum microneutralization assay against the SARS-CoV-2 Washington strain and the Delta variant. The animals vaccinated with the DNA vaccine followed by a boosting dose of the SARS-CoV-2 PsIV exhibited the highest neutralizing antibody responses and were able to quickly clear infection after an intranasal challenge with the SARS-CoV-2 Delta variant. Overall, the data show that the Advax-CpG-adjuvanted SARS-CoV-2 PsIV, either by itself or as a booster shot following nucleic acid (NA) vaccines, has the potential to protect against emerging variants.

Pseudovirus-Based Assays for the Measurement of Antibody-Mediated Neutralization of SARS-CoV-2.

SARS-CoV-2 has emerged as a significant cause of morbidity and mortality worldwide. Virus neutralization assays are critical for the development and evaluation of vaccines and immunotherapeutics, as well as for conducting basic research into the immune response, spread, and pathogenesis of this disease. However, neutralization assays traditionally require the use of infectious virus which must be carefully handled in a BSL-3 setting, thus complicating the assay and restricting its use to labs with access to BSL-3 facilities. Pseudovirus-based assays are an alternative to the use of infectious virus. SARS-CoV-2 pseudovirus contains only the spike structural protein, and infection results in a single round of replication, thus allowing for the assay to be run safely under BSL-2 conditions. In this chapter, we describe protocols and considerations for the production and titration of lentivirus-based SARS-CoV-2 pseudovirus, as well as for running and analysis of FACS-based pseudovirus neutralization assays.

Enhanced Immunogenicity of Inactivated Dengue Vaccines by Novel Polysaccharide-Based Adjuvants in Mice.

Dengue fever, caused by any of four dengue viruses (DENV1-4), is a major global burden. Currently, there is no effective vaccine that prevents infection in dengue naïve populations. We tested the ability of two novel adjuvants (Advax-PEI and Advax-2), using aluminum hydroxide (alum) as control, to enhance the immunogenicity of formalin- or psoralen-inactivated (PIV or PsIV) DENV2 vaccines in mice. Mice were vaccinated on days 0 and 30, and serum samples were collected on days 30, 60, 90, and 101. Neutralizing antibodies were determined by microneutralization (MN) assays, and the geometric mean 50% MN (MN50) titers were calculated. For the PIV groups, after one dose MN50 titers were higher in the novel adjuvant groups compared to the alum control, while MN50 titers were comparable between the adjuvant groups after the second dose. For the PsIV groups, both novel adjuvants induced higher MN50 titers than the alum control after the second dose. Spleen cells were collected on days 45 and 101 for enzyme-linked immunospot (ELISPOT) for IFNγ and IL4. Both PIV and PsIV groups elicited different degrees of IFNγ and IL4 responses. Overall, Advax-2 gave the best responses just ahead of Advax-PEI. Given Advax-2's extensive human experience in other vaccine applications, it will be pursued for further development.

Etiologies of Acute Undifferentiated Febrile Illnesses in and near Iquitos from 1993 to 1999 in the Amazon River Basin of Peru.

The objective of this study was to determine the etiology of febrile illnesses among patients from October 1, 1993 through September 30, 1999, in the urban community of Iquitos in the Amazon River Basin of Peru. Epidemiological and clinical data as well as blood samples were obtained from consenting patients at hospitals, health clinics and private residences. Samples were tested for arboviruses in cell cultures and for IgM and IgG antibodies by ELISA. Blood smears were examined for malaria, and sera were tested for antibodies to Leptospira spp. by ELISA and microscopic agglutination. Among 6,607 febrile patients studied, dengue viruses caused 14.6% of the cases, and Venezuelan equine encephalitis virus caused 2.5%, Oropouche virus 1.0%, Mayaro virus 0.4%, and other arboviruses caused 0.2% of the cases. Also, 22.9% of 4,844 patients tested were positive for malaria, and of 400 samples tested, 9% had evidence of acute leptospirosis. Although the study was not designed to assess the importance of these pathogens as a cause of human morbidity in the total population, these results indicate that arboviruses, leptospirosis, and malaria were the cause of approximately 50% of the febrile cases. Although the arboviruses that were diagnosed can produce asymptomatic infections, our findings increased the overall understanding of the relative health burden of these infections, as well as baseline knowledge needed for designing and implementing further studies to better assess the health impact and threat of these pathogens in the Amazon Basin of Peru.

Evidence for limited spatial spread in an exotic longhorn beetle, Tetropium fuscum (Coleoptera: Cerambycidae).

The longhorn beetle Tetropium fuscum F. (Coleoptera: Cerambycidae) has become established in Nova Scotia, Canada, where it coexists with Tetropium cinnamopterum Kirby. The two Tetropium species share a similar ecological niche and use the same volatile cues for mate attraction. Exotic T. fuscum was introduced near Halifax, Nova Scotia, in approximately 1990, but the rate of its spread 20 yr later has not been documented. We report a large-scale, 3-yr study that investigates the distribution of T. fuscum relative to its site of introduction. Traps baited with male-produced pheromone and host volatiles were used to estimate the relative abundance of the two Tetropium species. Adult T. fuscum emerged 1-2 wk earlier than T. cinnamopterum each year between 2008 and 2010. The spatial distribution of T. fuscum was characterized by a sharp decline in abundance in relation to its point of introduction, up to a threshold distance of approximately 80 km beyond which T. fuscum is rare in comparison with native T. cinnamopterum. The restricted range of T. fuscum 20 yr after its introduction may be attributed to limited dispersal of adults or reproductive failures of low-density populations. The distribution of T. fuscum seemed stable between 2008 and 2010. In 1 of 3 yr, the abundance of T. cinnamopterum increased with the distance to the site of introduction of T. fuscum, which suggests competitive interactions between the two Tetropium species.

Immunogenicity of Adjuvanted Psoralen-Inactivated SARS-CoV-2 Vaccines and SARS-CoV-2 Spike Protein DNA Vaccines in BALB/c Mice.

The development of a safe and effective vaccine to protect against COVID-19 is a global priority due to the current high SARS-CoV-2 infection rate. Currently, there are over 160 SARS-CoV-2 vaccine candidates at the clinical or pre-clinical stages of development. Of these, there are only three whole-virus vaccine candidates produced using ß-propiolactone or formalin inactivation. Here, we prepared a whole-virus SARS-CoV-2 vaccine (SARS-CoV-2 PsIV) using a novel psoralen inactivation method and evaluated its immunogenicity in mice using two different adjuvants, alum and Advax-2. We compared the immunogenicity of SARS-CoV-2 PsIV against SARS-CoV-2 DNA vaccines expressing either full-length or truncated spike proteins. We also compared the psoralen-inactivated vaccine against a DNA prime, psoralen-inactivated vaccine boost regimen. After two doses, the psoralen-inactivated vaccine, when administered with alum or Advax-2 adjuvants, generated a dose-dependent neutralizing antibody responses in mice. Overall, the pattern of cytokine ELISPOT responses to antigen-stimulation observed in this study indicates that SARS-CoV-2 PsIV with the alum adjuvant promotes a Th2-type response, while SARS-CoV-2 PsIV with the Advax-2 adjuvant promotes a Th1-type response.

Comparison of purified psoralen-inactivated and formalin-inactivated dengue vaccines in mice and nonhuman primates.

Dengue fever, caused by dengue viruses (DENV 1-4) is a leading cause of illness and death in the tropics and subtropics. Therefore, an effective vaccine is urgently needed. Currently, the only available licensed dengue vaccine is a chimeric live attenuated vaccine that shows varying efficacy depending on serotype, age and baseline DENV serostatus. Accordingly, a dengue vaccine that is effective in seronegative adults, children of all ages and in immunocompromised individuals is still needed. We are currently researching the use of psoralen to develop an inactivated tetravalent dengue vaccine. Unlike traditional formalin inactivation, psoralen inactivates pathogens at the nucleic acid level, potentially preserving envelope protein epitopes important for protective anti-dengue immune responses. We prepared highly purified monovalent vaccine lots of formalin- and psoralen-inactivated DENV 1-4, using Capto DeVirS and Capto Core 700 resin based column chromatography. Tetravalent psoralen-inactivated vaccines (PsIV) and formalin-inactivated vaccines (FIV) were prepared by combining the four monovalent vaccines. Mice were immunized with either a low or high dose of PsIV or FIV to evaluate the immunogenicity of monovalent as well as tetravalent formulations of each inactivation method. In general, the monovalent and tetravalent PsIVs elicited equivalent or higher titers of neutralizing antibodies to DENV than the FIV dengue vaccines and this response was dose dependent. The immunogenicity of tetravalent dengue PsIVs and FIVs were also evaluated in nonhuman primates (NHPs). Consistent with what was observed in mice, significantly higher neutralizing antibody titers for each dengue serotype were observed in the NHPs vaccinated with the tetravalent dengue PsIV compared to those vaccinated with the tetravalent dengue FIV, indicative of the importance of envelope protein epitope preservation during psoralen inactivation of DENV.

Tetravalent dengue DNA vaccine is not immunogenic when delivered by retrograde infusion into salivary glands.

INTRODUCTION AND BACKGROUND: A tetravalent DNA vaccine for Dengue virus is under development but has not yet achieved optimal immunogenicity. Salivary glands vaccination has been reported efficacious in rodents and dogs. We report on a pilot study testing the salivary gland as a platform for a Dengue DNA vaccine in a non-human primate model. MATERIALS AND METHODS: Four cynomolgus macaques were used in this study. Each macaque was pre-medicated with atropine and sedated with ketamine. Stensen's duct papilla was cannulated with a P10 polyethylene tube, linked to a 500ul syringe. On the first two infusions, all macaques were infused with 300ul of TVDV mixed with 2 mg of zinc. For the 3rd infusion, to increase transfection into salivary tissue, two animals received 100uL TVDV mixed with 400uL polyethylenimine 1µg/ml (PEI) and the other two animals received 500uL TVDV with zinc. Antibody titers were assessed 4 weeks following the second and third infusion. RESULTS AND CONCLUSIONS: SGRI through Stensen's duct is a well-tolerated, simple and easy to reproduce procedure. TVDV infused into macaques salivary glands elicited a significantly weaker antibody response than with different delivery methods.

Role of dendritic cells in antibody-dependent enhancement of dengue virus infection.

Dengue viruses (DV), composed of four distinct serotypes (DV1 to DV4), cause 50 to 100 million infections annually. Durable homotypic immunity follows infection but may predispose to severe subsequent heterotypic infections, a risk conferred in part by the immune response itself. Antibody-dependent enhancement (ADE), a process best described in vitro, is epidemiologically linked to complicated DV infections, especially in Southeast Asia. Here we report for the first time the ADE phenomenon in primary human dendritic cells (DC), early targets of DV infection, and human cell lines bearing Fc receptors. We show that ADE is inversely correlated with surface expression of DC-SIGN (DC-specific intercellular adhesion molecule-3-grabbing nonintegrin) and requires Fc gamma receptor IIa (FcgammaRIIa). Mature DC exhibited ADE, whereas immature DC, expressing higher levels of DC-SIGN and similar FcgammaRIIa levels, did not undergo ADE. ADE results in increased intracellular de novo DV protein synthesis, increased viral RNA production and release, and increased infectivity of the supernatants in mature DC. Interestingly, tumor necrosis factor alpha and interleukin-6 (IL-6), but not IL-10 and gamma interferon, were released in the presence of dengue patient sera but generally only at enhancement titers, suggesting a signaling component of ADE. FcgammaRIIa inhibition with monoclonal antibodies abrogated ADE and associated downstream consequences. DV versatility in entry routes (FcgammaRIIa or DC-SIGN) in mature DC broadens target options and suggests additional ways for DC to contribute to the pathogenesis of severe DV infection. Studying the cellular targets of DV infection and their susceptibility to ADE will aid our understanding of complex disease and contribute to the field of vaccine development.

A tetravalent dengue vaccine based on a complex adenovirus vector provides significant protection in rhesus monkeys against all four serotypes of dengue virus.

Nearly a third of the human population is at risk of infection with the four serotypes of dengue viruses, and it is estimated that more than 100 million infections occur each year. A licensed vaccine for dengue viruses has become a global health priority. A major challenge to developing a dengue vaccine is the necessity to produce fairly uniform protective immune responses to all four dengue virus serotypes. We have developed two bivalent dengue virus vaccines, using a complex adenovirus vector, by incorporating the genes expressing premembrane (prM) and envelope (E) proteins of dengue virus types 1 and 2 (dengue-1 and -2, respectively) (CAdVax-Den12) or dengue-3 and -4 (CAdVax-Den34). Rhesus macaques were vaccinated by intramuscular inoculation of a tetravalent dengue vaccine formulated by combining the two bivalent vaccine constructs. Vaccinated animals produced high-titer antibodies that neutralized all four serotypes of dengue viruses in vitro. The ability of the vaccine to induce rapid, as well as sustained, protective immune responses was examined with two separate live-virus challenges administered at 4 and 24 weeks after the final vaccination. For both of these virus challenge studies, significant protection from viremia was demonstrated for all four dengue virus serotypes in vaccinated animals. Viremia from dengue-1 and dengue-3 challenges was completely blocked, whereas viremia from dengue-2 and dengue-4 was significantly reduced, as well as delayed, compared to that of control-vaccinated animals. These results demonstrate that the tetravalent dengue vaccine formulation provides significant protection in rhesus macaques against challenge with all four dengue virus serotypes.

Advances in dengue vaccine development.

Dengue viruses are the most important arboviruses causing human disease. Expansion of the disease in recent decades to include more geographical areas of the world, an appreciation of the disease burden and market potentials have spurred a flurry of activity in the development of vaccines to combat dengue viruses. Recent progress in this area and some of the obstacles associated with this development are discussed.

Enhanced immunogenicity and protective efficacy of a tetravalent dengue DNA vaccine using electroporation and intradermal delivery.

Phase 1 clinical trials with a DNA vaccine for dengue demonstrated that the vaccine is safe and well tolerated, however it produced less than optimal humoral immune responses. To determine if the immunogenicity of the tetravalent dengue DNA vaccine could be enhanced, we explored alternate, yet to be tested, methods of vaccine administration in non-human primates. Animals were vaccinated on days 0, 28 and 91 with either a low (1 mg) or high (5 mg) dose of vaccine by the intradermal or intramuscular route, using either needle-free injection or electroporation devices. Neutralizing antibody, IFN-γ T cell and memory B cell responses were compared to a high dose group vaccinated with a needle-free intramuscular injection delivery device similar to what had been used in previous preclinical and clinical studies. All previously untested vaccination methodologies elicited improved immune responses compared to the high dose needle-free intramuscular injection delivery group. The highest neutralizing antibody responses were observed in the group that was vaccinated with the high dose formulation via intradermal electroporation. The highest IFN-γ T cell responses were also observed in the high dose intradermal electroporation group and the CD8+ T cells were the dominant contributors for the IFNγ response. Memory B cells were detected for all four serotypes. More than a year after vaccination, groups were challenged with dengue-1 virus. Both the low and high dose intradermal electroporation groups had significantly fewer days of dengue-1 virus RNAemia compared to the control group. The results from this study demonstrate that using either an electroporation device and/or the intradermal route of delivery increases the immune response generated by this vaccine in non-human primates and should be explored in humans.

Safety and Immunogenicity of a Tetravalent Dengue DNA Vaccine Administered with a Cationic Lipid-Based Adjuvant in a Phase 1 Clinical Trial.

We conducted an open label, dose escalation Phase 1 clinical trial of a tetravalent dengue DNA vaccine (TVDV) formulated in Vaxfectin® to assess safety and immunogenicity. A total of 40 dengue- and flavivirus-naive volunteers received either low-dose (1 mg) TVDV alone (N = 10, group 1), low-dose TVDV (1 mg) formulated in Vaxfectin (N = 10, group 2), or high-dose TVDV (2 mg, group 3) formulated in Vaxfectin® (N = 20). Subjects were immunized intramuscularly with three doses on a 0-, 30-, 90-day schedule and monitored. Blood samples were obtained after each immunization and various time points thereafter to assess anti-dengue antibody and interferon gamma (IFNγ) T-cell immune responses. The most common adverse events (AEs) across all groups included mild to moderate pain and tenderness at the injection site, which typically resolved within 7 days. Common solicited signs and symptoms included fatigue (42.5%), headache (45%), and myalgias (47.5%). There were no serious AEs related to the vaccine or study procedures. No anti-dengue antibody responses were detected in group 1 subjects who received all three immunizations. There were minimal enzyme-linked immunosorbent assay and neutralizing antibody responses among groups 2 and 3 subjects who completed the immunization schedule. By contrast, IFNγ T-cell responses, regardless of serotype specificity, occurred in 70%, 50%, and 79% of subjects in groups 1, 2, and 3, respectively. The largest IFNγ T-cell responses were among group 3 subjects. We conclude that TVDV was safe and well-tolerated and elicited predominately anti-dengue T-cell IFNγ responses in a dose-related fashion.

Induction of bivalent immune responses by expression of dengue virus type 1 and type 2 antigens from a single complex adenoviral vector.

There are approximately 100 million new cases of dengue (DEN) virus infection each year. Infection can result in illness ranging from a mild fever to hemorrhaging, shock, or even death. There are four serotypes of dengue virus (DEN1-4), and immunity to one serotype does not cross protect from infection with other serotypes. Currently there are no approved vaccines for dengue fever. In this report, we describe the construction of a bivalent dengue virus vaccine using a complex recombinant adenovirus approach to express multiple genes of DEN1 and DEN2 serotypes. In vaccinated mice, this vector induced humoral immune responses against all four dengue serotypes as measured by enzyme-linked immunosorbent assay. However, the neutralizing antibody responses were specific for DEN1 and DEN2 serotypes. Expansion of this vaccine development platform towards the DEN3 and DEN4 serotypes can lead towards the development of an adenovirus-based tetravalent dengue vaccine.

NK cell degranulation as a marker for measuring antibody-dependent cytotoxicity in neutralizing and non-neutralizing human sera from dengue patients.

The study assessed antibody-dependent NK cell degranulation, a biomarker relevant to antibody-dependent cell cytotoxicity (ADCC), to analyze dengue immune sera. We first determined binding intensity of patient sera to the surface of DENV-infected cells and examined the types of antigens expressed on infected cells. Antigens from pre-membrane (PreM) and envelope (E), but not from NS proteins were detected on the surface of infected cells. After adding NK cells to infected target cells previously treated with patient sera, rapid NK cell degranulation was observed. Non-neutralizing patient sera generated comparable NK cell degranulation as that of neutralizing sera, suggesting ADCC may be a protective mechanism apart from Ab neutralization. The level of NK cell degranulation varied dramatically among human individuals and was associated with the level of CD16 expression on NK cells, informing on the complexity of ADCC among human population.

An immunocytometric assay based on dengue infection via DC-SIGN permits rapid measurement of anti-dengue neutralizing antibodies.

Dengue remains a global public health threat and development of a safe and effective vaccine is a principal public health goal. The primary correlate of immunity is thought to be neutralizing antibodies. Currently, the plaque reduction neutralization test (PRNT) is the gold standard measure of dengue neutralizing antibody responses, but this test is limited by time-consuming performance. In addition, some feel that use of viral strains adapted to grow in Vero or BHK cells may not accurately reflect protective responses. A human cell line transfected to express a putative natural dengue receptor, DC-SIGN (CD209), was used to measure antibody-mediated dengue neutralization. Using neutralizing monoclonal antibodies, immune sera, and laboratory adapted dengue viruses, serotype-specific neutralizing activity was demonstrated similar to that seen in the Vero PRNT. Importantly, serotype-specific neutralizing activity against recently isolated dengue strains with less heterotypic cross-neutralization than laboratory adapted viruses was also demonstrated.

Four dengue virus serotypes found circulating during an outbreak of dengue fever and dengue haemorrhagic fever in Jakarta, Indonesia, during 2004.

Periodic outbreaks of dengue have emerged in Indonesia since 1968, with the severity of resulting disease increasing in subsequent years. In early 2004, a purported dengue outbreak erupted across the archipelago, with over 50,000 cases and 603 deaths reported. To confirm the disease aetiology and to provide an epidemiological framework of this epidemic, an investigation was conducted in ten hospitals within the capital city of Jakarta. Clinical and laboratory findings were determined from a cohort of 272 hospitalised patients. Exposure to dengue virus was determined in 180 (66.2%) patients. When clinically assessed, 100 (55.6%) of the 180 patients were classified as having dengue fever (DF), 31 (17.2%) as DF with haemorrhagic manifestations and 49 (27.2%) as dengue haemorrhagic fever (DHF). Evidence from haemagglutination inhibition assays suggested that 33/40 (82.5%) of those with DHF from which laboratory evidence was available suffered from a secondary dengue infection. All four dengue viruses were identified upon viral isolation, with DEN-3 being the most predominant serotype recovered, followed by DEN-4, DEN-2 and DEN-1. In summary, the 2004 outbreak of dengue in Jakarta, Indonesia, was characterised by the circulation of multiple virus serotypes and resulted in a relatively high percentage of a representative population of hospitalised patients developing DHF.

Needle-free injection of DNA vaccines: a brief overview and methodology.

The development of needle-free injection originally stemmed from a general apprehension of needle injections, disease transmission by accidental needle-sticks, and the need for effective mass immunization. Naked DNA vaccines, as attractive and universal as they appear, have not produced robust immune responses in test systems. However, proof of principle for DNA vaccines has been validated with a number of vaccine candidates in a variety of test systems, and the concept of DNA vaccines as a generic platform for vaccines still remains viable and attractive. Many avenues are being explored to enhance the immunogenicity of DNA vaccines. The easiest and most straightforward approach that can be quickly transitioned to a clinical trial setting is vaccine delivery by a needle-free jet injector. This approach has shown much potential in a number of cases and should become the lead method for enhancing DNA vaccines. This approach requires no additional development, and with an expanding market and willingness from jet injector manufacturers to produce prefilled syringes, the technique should become feasible for larger phase II/phase III trials.