Formalin Inactivation Research Articles

Assessment of Different Newcastle Disease Virus Antigens and Inactivators of Binary Ethylene Amine and Formalin for the Hemagglutination Inhibition Assay.

Newcastle disease is a severe viral threat to the global poultry industry due to its high prevalence and rapid transmission. Evaluating vaccination timing and effectiveness is crucial, often accomplished through the hemagglutination inhibition (HI) assay. This test relies on the virus's agglutination ability in certain animals, utilizing various inactivated antigens. Our study aimed to assess multiple Newcastle viral antigens ( LaSota, clone, thermo-resistant strain, B1, and V4 ) inactivated by binary ethylene amine (BEA) and formalin, seeking the best antigen and inactivator for the HI assay. We prepared the different ND antigens include; LaSota, Clone, thermo resistant, B1, V4 and the mixture of the antigens then inactivated them using BEA and formalin. The hemagglutination (HA) assay determined mean titers, comparing BEA and formalin inactivation. These antigens were also subjected to the HI test using 112 serum samples from different commercial poultry flocks to assess their performance. BEA-inactivated antigens exhibited significantly higher mean titers in the HA assay than formalin-inactivated antigens. In the evaluation of different antigens in the HI test, the mean titer of antigen B1 followed by clone and LaSota displayed a higher mean titer than others. In conclusion, this study recommends using Hitchner pathotype antigens, specifically the B1 vaccine, for Newcastle disease HI testing. BEA is the preferred inactivator, preserving antigen structure particularly the structure of hemagglutinin antigen while minimizing risks. These findings can enhance serological testing accuracy, contributing to more effective disease control and prevention in the poultry industry.

Effect of formalin percentage, incubation time and temperature on Clostridium chauvoei culture inactivation and immunogenicity

ObjectivesIn order to find the optimal inactivation conditions for Clostridium chauvoei culture, different factors were investigated and the immunogenicity of inactivated cultures was studied. MethodsC. chauvoei was cultured with different formalin percentages (0.3, 0.5 or 0.7% V/V), inactivation temperatures (37 °C or room temperature) and incubation times (one or two weeks). Sterility tests were performed and residual formaldehyde and pH were measured. Rabbits were immunized twice with inactivated cultures and sera were used for detection of immune response. ResultsIn the one-week experiment, 0.5 and 0.7% formalin inactivated the bacteria after one week, and the percentage of 0.3 inactivated after three weeks. The residual formaldehyde at weeks 1 and 8 was not significantly different. In the two-week experiment, cultures treated with 0.3 and 0.5% formalin were inactivated after four weeks, and those with 0.7% formalin were inactivated after three weeks. Residual formaldehyde at week 8 differed significantly from that of week 1. Residual formaldehyde was affected by incubation temperature since it was lower at 37 °C than in room temperature. Also, a significant effect was observed for formalin on pH, as higher formalin contents led to lower pH values of the cultures. ELISA showed the lowest antibody titer achieved by 0.7% formalin group. Antibody titer was not different between 0.3 and 0.5% formalin. ConclusionsThe best condition for inactivation of C. chauvoei was considered as one-week incubation with 0.5% formalin at 37 °C, leading to a high antibody response.

Efficacy of bivalent vaccine against Aeromonas salmonicida and Edwardsiella tarda infections in turbot.

In recent years, more than one pathogenic organism has usually been isolated from diseased turbot Scophthalmus maximus, creating a pressing need for the development of combination vaccines to prevent fish diseases brought on simultaneously by various infections. In this study, the inactivated bivalent vaccine of Aeromonas salmonicida and Edwardsiella tarda was prepared by the formalin inactivation method. After challenge with A. salmonicida and E. tarda at 4 weeks post-vaccination in turbot, the relative percentage survival (RPS) of the inactivated bivalent vaccine was 77.1%. In addition, we assessed the effects of the inactivated bivalent vaccine and evaluated the immunological processes after immunization in a turbot model. Serum antibody titer and lysozyme activity of the vaccinated group were both upregulated and higher than that in control group after vaccination. The expression levels of genes (TLR2, IL-1β, CD4, MHCI, MHCⅡ) that related to antigen recognition, processing and presentation were also studied in the liver, spleen and kidney tissues of vaccinated turbot. All the detected genes in the vaccinated group had a significant upward trend, and most of them reached the maximum value at 3-4 weeks, which had significant differences from the control group, suggesting that antigen recognition, processing and presentation pathway was activated by the inactivated bivalent vaccine. Our study provides a basis for further application of the killed bivalent vaccine against A. salmonicida and E. tarda in turbot, making it good potential that can be applied in aquaculture.

Evaluation of Clostridium septicum hemolytic activity, administration route, and dosage volume of a clostridial dermatitis (cellulitis) bacterin-toxoid on humoral immune response in commercial turkeys

Clostridial cellulitis or dermatitis affects commercial turkey flocks, primarily as they approach market age. In the field, this disease has been effectively controlled with antibiotics, but alternatives to antibiotics are needed. Bacterin-toxoid vaccination programs have been shown to prevent clostridial diseases in other species, including humans. Results from previous field studies indicate that vaccination with an experimental whole-cell Clostridium septicum (CS) bacterin-toxoid oil emulsion vaccine reduced clostridial dermatitis-associated mortality and antibiotic usage for some commercial turkey flocks, but vaccination was not always efficacious. To improve vaccine efficacy, studies were conducted to optimize the antigenic component of the experimental vaccine and to determine the appropriate antigen to adjuvant ratio, route, and volume for vaccine administration. It was determined that the phase of culture at time of formalin inactivation played a key role in serum antibody titer and larger volume vaccine doses produced higher serum antibody immune response regardless of antigen:adjuvant formulation ratio or route of injection. No significant differences (P > 0.05) were found between formulation ratios or between the subcutaneous and tail head injection sites. Based on these results, we propose to look further into the relationship between culture phase and antigenic components produced by CS under different culture conditions.

Gamma Irradiation-Inactivated Respiratory Syncytial Virus Vaccine Provides Protection but Exacerbates Pulmonary Inflammation by Switching from Prefusion to Postfusion F Protein.

Respiratory syncytial virus (RSV) is a common respiratory pathogen that causes lower respiratory diseases among infants and elderly people. Moreover, formalin-inactivated RSV (FI-RSV) vaccine induces serious enhanced respiratory disease (ERD). Radiation has been investigated as an alternative approach for producing inactivated or live-attenuated vaccines, which enhance the antigenicity and heterogeneous protective effects of vaccines compared with conventional formalin inactivation. In this study, we developed an RSV vaccine using gamma irradiation and analyzed its efficacy against RSV vaccine-induced ERD in a mouse model. Although gamma irradiation-inactivated RSV (RI-RSV) carbonylation was lower than FI-RSV carbonylation and RI-RSV showed a significant antibody production and viral clearance, RI-RSV caused more obvious body weight loss, pulmonary eosinophil infiltration, and pulmonary mucus secretion. Further, the conversion of prefusion F (pre-F) to postfusion F (post-F) was significant for both RI-RSV and FI-RSV, while that of RI-RSV was significantly higher than that of FI-RSV. We found that the conversion from pre- to post-F during radiation was caused by radiation-induced reactive oxygen species. Although we could not propose an effective RSV vaccine manufacturing method, we found that ERD was induced by RSV vaccine by various biochemical effects that affect antigen modification during RSV vaccine manufacturing, rather than simply by the combination of formalin and alum. Therefore, these biochemical actions should be considered in future developments of RSV vaccine. IMPORTANCE Radiation inactivation for viral vaccine production has been known to elicit a better immune response than other inactivation methods due to less surface protein damage. However, we found in this study that radiation-inactivated RSV (RI-RSV) vaccine induced a level of immune response similar to that induced by formalin-inactivated RSV (FI-RSV). Although RI-RSV vaccine showed less carbonylation than FI-RSV, it induced more conformational changes from pre-F to post-F due to the gamma radiation-induced reactive oxygen species response, which may be a key factor in RI-RSV-induced ERD. Therefore, ERD induced by RSV vaccine may be due to pre-F to post-F denaturation by random protein modifications caused by external stress. Our findings provide new ideas for inactivated vaccines for RSV and other viruses and confirm the importance of pre-F in RSV vaccines.

Formalin Inactivation of Virus for Safe Downstream Processing of Routine Stool Parasite Examination during the COVID-19 Pandemic.

During the COVID-19 pandemic, the parasitology laboratories dealing with fecal samples for the diagnosis of gastrointestinal parasitic infections are confronting the unsaved virus-containing samples. To allow for safe downstream processing of the fecal samples, a protocol for preparing a fecal smear is urgently needed. Formalin was tested with or without isotonic forms for virus inactivation using porcine epidemic diarrhea virus (PEDV) as a representative, as it belongs to the Coronaviridae family. The results revealed complete inactivation activity of 10% formalin and 10% isotonic formalin on coronavirus after 5 min of treatment at room temperature. Both also inhibited Naegleria fowleri growth after 5 min of treatment at 37 °C without disruption of the structure. In addition to these key findings, it was also found that isotonic formalin could stabilize both red and white blood cells when used as a solution to prepare fecal smears comparable to the standard method, highlighting its value for use instead of 0.9% normal saline solution for the quantification of blood cells without active virus. The 10% isotonic formalin is useful to safely prepare a fecal smear for the diagnosis of parasites and other infections of the gastrointestinal tract during the COVID-19 pandemic.

Protective effects of immersion immunization of koi with Escherichia coli DH5α carrying DNA vaccine against koi herpesvirus

This study aims to immunize the koi fingerlings immersion using the formalin-killed and freeze-dried E. coli DH5α carrying plasmid for the KHV DNA vaccine. 200 fish on each tank in a total water volume of 20 L. Each tanks consists of different vaccination group: PBS as control (10 mL; C), empty E. coli DHα (10 mL at 108 CFU mL-1; E), formalinkilled E. coli DHα:ORF81 (10 mL at 108 CFU mL-1; KE), freezedried E.coli DHα:ORF81 without formalin inactivation (10 mL at 108 CFU mL-1; FE), and formalin-killed and then freeze-dried E. coli DHα:ORF81 (10 mL at 108 CFU mL-1; KFE). The bath vaccination was conducted for 1 × 30 min. For the challenged test, fish were immersed with the 100 mL of LD50 dose of KHV (10–2 dilution from the KHV stock) for 30 min. The vaccination using E. coli DH5α:ORF81 could significantly modulate the innate and adaptive immunity of the fish and result in higher fish survival after KHV infection. The vaccination using formalin-killed or formalin-killed and freezedried E. coli DH5α:ORF81 will be further developed as an alternative to the costefficient koi or carp fingerlings vaccination method.

Improved Whole Gamma Irradiated Avian Influenza Subtype H9N2 Virus Vaccine Using Trehalose and Optimization of Vaccination Regime on Broiler Chicken

Gamma (γ)-radiation can target viral genome replication and preserve viral structural proteins compared to formalin inactivation. Thus, a stronger immunity could be induced after the inoculation of the irradiated virus. In this study, γ-irradiated low-pathogenic avian influenza virus-H9N2 (LPAIV-H9N2) was used to immunize the broiler chicken in two formulations, including γ-irradiated LPAIV-H9N2 with 20% Trehalose intranasally (IVT.IN) or γ-irradiated LPAIV-H9N2 plus Montanide oil adjuvant ISA70 subcutaneously (IV+ISA.SC) in comparison with formalin-inactivated LPAIV-H9N2 vaccine intranasally (FV.IN) or formalin-inactivated LPAIV-H9N2 plus ISA70 subcutaneously (FV+ISA.SC). Two vaccination regimes were employed; the first one was primed on day 1 and boosted on day 15 (early regime), and the second one was primed on day 11 and boosted on day 25 (late regime). A challenge test was performed with a live homologous subtype virus. Virus shedding was monitored by quantifying the viral load via RT-qPCR on tracheal and cloacal swabs. Hemagglutination inhibition (HI) antibody titration and stimulation index (SI) of the splenic lymphocyte proliferation were measured, respectively, by HI test and Cell Proliferation assay. Cytokine assay was conducted by the RT-qPCR on antigen-stimulated spleen cells. The results of the HI test showed significant increases in antibody titer in all vaccinated groups, but it was more evident in the IVT late vaccination regime, reaching 5.33 log2. The proliferation of stimulated spleen lymphocytes was upregulated more in the IVT.IN vaccine compared to other vaccines. The mRNA transcription levels of T-helper type 1 cytokines such as interferon-gamma (IFN-γ) and interleukin 2 (IL-2) were upregulated in all vaccinated groups at the late regime. Moreover, IL-6, a pro-inflammatory cytokine was upregulated as well. However, upregulation was more noticeable in the early vaccination than the late vaccination (p< 0.05). After the challenge, the monitoring of virus shedding for the H9 gene represented an extremely low viral load. The body weight loss was not significant (p > 0.05) among the vaccinated groups. In addition, the viral load of <100.5 TCID50/ml in the vaccinated chicken indicated the protective response for all the vaccines. Accordingly, the IVT vaccine is a good candidate for the immunization of broiler chicken via the intranasal route at late regime.

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 β-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.

Differential protection of Mayaro virus antigen-antibody binding by various inactivation approaches: Implication in vaccine development

Abstract Mayaro virus (MAYV) is an emerging alphavirus in the family Togaviridae that causes a self-limited febrile illness accompanied by maculopapular rash and arthralgia. MAYV is reported throughout South America and the Caribbean and has potential to cause widespread outbreaks. There is no FDA-approved vaccine for MAYV. The overall objectives of this study were to inactivate MAYV utilizing various inactivation approaches, and to identify a highly safe vaccine candidate with optimum immunogenicity. MAYV was inactivated by three different mechanisms: formalin; 1, 5 iodonaphthyl azide (INA); and γ-radiation. Formalin inactivation (F-iMAYV) was performed with 0.05 or 0.1% formalin at 35°C. INA-inactivation (INA-iMAYV) was performed with 10, 50, or 100 μM INA combined with UV exposure. Lastly, MAYV was inactivated with 10, 20, or 30 kGy of γ-radiation in the presence of a protein-protecting manganese-decapeptide-phosphate complex (MDP-iMAYV). MAYV was inactivated by all the three approaches. Dose- and incubation time-dependent effects were observed with formalin inactivation. 0.1% formalin with an incubation time of 48h inactivated MAYV; however, further evaluation is ongoing. MAYV was completely inactivated with &amp;gt;50 μM dose of INA and by all three doses of γ-radiation. Differential protection of MAYV epitopes was observed after inactivation with three agents and with different doses of the inactivating agent. The most consistent antigen (Ag)-antibody (Ab) binding was observed in INA-iMAYV, followed by MDP-iMAYV. Ag-Ab binding was almost completely lost in F-iMAYV. The data suggests that INA and MDP-inactivated MAYV can be further studied as vaccine candidates. Studies are planned to evaluate immunogenicity in-vivo.

Virulence pattern of circulating aeromonads isolated from farmed Nile tilapia in Tanzania and novel antibiotic free attenuation of Aeromonas hydrophila strain TZR7-2018

Aeromonads are gram-negative, rod-shaped, facultative anaerobes bacteria known to cause motile aeromonads septicemia diseases (MAS) in warm freshwater farmed fish. Outbreaks are associated with pathogenicity of aeromonads in fish which is partly contributed by virulence characteristics of the etiological agent. The objective of this study was to assess the virulence characteristics of the previously isolated and identified aeromonads, and attenuate potential Aeromonas hydrophila strain TZR7-2018 to serve as local vaccine candidate. Six virulence genes and other virulence characteristics were molecularly and phenotypically assessed both using in-vitro and in-vivo approaches. Attenuation of A. hydrophila parent strain TZR7-2018+ was performed by passaging through thermal continuous sub-culturing 40 times in Tryptic soy agar (TSA). Bacterin was prepared by formalin inactivation from the same parent strain. Humoral responses were assayed using quantitative serological agglutination test (qSAT) while protective efficacy was measured through relative percent survival (RPS). A total 240 Nile tilapia fingerlings with an average weight of 8.1 ± 0.4 g were used in all in-vivo studies. The presence of aerolysin (aer), cytotoxic enterotoxin (act), elastase (ahy), haemolysin (hly), serine (ser) and polar flagella (fla) genes were determined using PCR. Out of 201 isolates, 75.1 % (151/201) of the aeromonads possessed virulence genes (120=A. hydrophila and 31=Aeromonas veronii). The virulence gene pattern of aer/hly/fla was the most prominent with the prevalence of 12.6 %. The attenuated strain TZR7-2018− showed reduced: colon size, multiplication rate, cell size and loss in; haemolysis, motility and capsule. Humoral responses increased gradually and reached maximum at day 28 in both attenuated and bacterin formulation given through intraperitoneal (IP) injection and immersion (IM). A RPS of 82.3 %, 71.4 % and 85.1 %, were recorded to the attenuated vaccine given through IP and IM and bacterin provided through IP respectively.Therefore the attenuated strain TZR7-2018− obtained through thermal continuous subculture technique and the bacterin proved to be efficacious and can serve as vaccine candidate.

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.

A comparative study of the effect of UV and formalin inactivation on the stability and immunogenicity of a Coxsackievirus B1 vaccine

Type B Coxsackieviruses (CVBs) belong to the enterovirus genus, and they cause both acute and chronic diseases in humans. CVB infections usually lead to flu-like symptoms but can also result in more serious diseases such as myocarditis, aseptic meningitis and life-threatening multi-organ infections in young infants. Thus, CVBs have long been considered as important targets of future vaccines.We have previously observed CVB1 capsid disintegration and virus concentration decrease with 12-day long formalin inactivation protocol. Here a scalable ion exchange chromatography purification method was developed, and purified CVB1 was inactivated with UV-C or formalin. Virus morphology and concentration remained unchanged, when the UV (2 min) or formalin (5 days) inactivation were performed in the presence of tween80 detergent. The concentration of the native and UV inactivated CVB1 remained constant at 4 °C during a six months stability study, whereas the concentration of the formalin inactivated vaccine decreased 29% during this time. UV treatment decreased, whereas formalin treatment increased the thermal stability of the capsid.The formalin inactivated CVB1 vaccine was more immunogenic than the UV inactivated vaccine; the protective neutralizing antibody levels were higher in mice immunized with formalin inactivated vaccine. High levels of CVB1 neutralizing antibodies as well as IgG1 antibodies were detected in mice that were protected against viremia induced by experimental CVB1 infection.In conclusion, this study describes a scalable ion exchange chromatography purification method and optimized 5-day long formalin inactivation method that preserves CVB1 capsid structure and immunogenicity. Formalin treatment stabilizes the virus particle at elevated temperatures, and the formalin inactivated vaccine induces high levels of serum IgG1 antibodies (Th2 type response) and protective levels of neutralizing antibodies. Formalin inactivated CVB vaccines are promising candidates for human clinical trials.

Optimized production and purification of Coxsackievirus B1 vaccine and its preclinical evaluation in a mouse model

Coxsackie B viruses are among the most common enteroviruses, causing a wide range of diseases. Recent studies have also suggested that they may contribute to the development of type 1 diabetes. Vaccination would provide an effective way to prevent CVB infections, and the objective of this study was to develop an efficient vaccine production protocol for the generation of novel CVB vaccines. Various steps in the production of a formalin-inactivated Coxsackievirus B1 (CVB1) vaccine were optimized including the Multiplicity Of Infection (MOI) used for virus amplification, virus cultivation time, type of cell growth medium, virus purification method and formulation of the purified virus. Safety and immunogenicity of the formalin inactivated CVB1 vaccine was characterized in a mouse model. Two of the developed methods were found to be optimal for virus purification: the first employed PEG-precipitation followed by gelatin-chromatography and sucrose cushion pelleting (three-step protocol), yielding 19-fold increase in virus concentration (0.06µg/cm2) as compared to gold standard method. The second method utilized tandem sucrose pelleting without a PEG precipitation step, yielding 83-fold increase in virus concentration (0.24µg/cm2), but it was more labor-intensive and cannot be efficiently scaled up. Both protocols provide radically higher virus yields compared with traditional virus purification protocols involving PEG-precipitation and sucrose gradient ultracentrifugation. Formalin inactivation of CVB1 produced a vaccine that induced a strong, virus-neutralizing antibody response in vaccinated mice, which protected against challenge with CVB1 virus. Altogether, these results provide valuable information for the development of new enterovirus vaccines.

Pre-fusion F is absent on the surface of formalin-inactivated respiratory syncytial virus.

The lack of a licensed vaccine for respiratory syncytial virus (RSV) can be partly attributed to regulatory hurdles resulting from vaccine enhanced respiratory disease (ERD) subsequent to natural RSV infection that was observed in clinical trials of formalin-inactivated RSV (FI-RSV) in antigen-naïve infants. To develop an effective vaccine that does not enhance RSV illness, it is important to understand how formalin and heat inactivation affected the antigenicity and immunogenicity of FI-RSV compared to native virus. Informed by atomic structures of RSV fusion (F) glycoprotein in prefusion (pre-F) and postfusion (post-F) conformations, we demonstrate that FI-RSV predominately presents post-F on the virion surface, whereas infectious RSV presents both pre-F and post-F conformations. This significant antigenic distinction has not been previously appreciated. Thus, a stabilized pre-F antigen is more representative of live RSV than F in its post-F conformation, as displayed on the surface of FI-RSV. This finding has major implications for discriminating current pre-F-based immunogens from FI-RSV used in historical vaccine trials.

Use of formalin in intensive aquaculture: properties, application and effects on fish and water quality

AbstractFormalin (aqueous solution of formaldehyde stabilized with methanol) is one of the most used disinfectants in aquaculture. It is used to eliminate infectious agents but may be responsible for negative effects on fish and water quality. This work focuses on physical–chemical properties of formalin and on its reactions and its use in intensive aquaculture and in environment. The types of treatments and the action mode of formalin are also described. The main consequences of formalin exposure to fish are damage in gills and alterations in mucous cells. Formalin also interacts with some treatments adopted in aquaculture establishments, for example biological filter, where the nitrification process seems to be strongly affected. With respect to formalin inactivation, formaldehyde (effluent) may be diluted with water or specific treatments should be conducted to decrease the concentration levels before its discharge into the environment. The common methodologies applied to aquaculture are not completely efficient on formalin removal from water, and the advanced oxidation processes may be a good alternative.

Formalin Inactivation of Japanese Encephalitis Virus Vaccine Alters the Antigenicity and Immunogenicity of a Neutralization Epitope in Envelope Protein Domain III.

Formalin-inactivated Japanese encephalitis virus (JEV) vaccines are widely available, but the effects of formalin inactivation on the antigenic structure of JEV and the profile of antibodies elicited after vaccination are not well understood. We used a panel of monoclonal antibodies (MAbs) to map the antigenic structure of live JEV virus, untreated control virus (UCV), formalin-inactivated commercial vaccine (FICV), and formalin-inactivated virus (FIV). The binding activity of T16 MAb against Nakayama-derived FICV and several strains of FIV was significantly lower compared to live virus and UCV. T16 MAb, a weakly neutralizing JEV serocomplex antibody, was found to inhibit JEV infection at the post-attachment step. The T16 epitope was mapped to amino acids 329, 331, and 389 within domain III (EDIII) of the envelope (E) glycoprotein. When we explored the effect of formalin inactivation on the immunogenicity of JEV, we found that Nakayama-derived FICV, FIV, and UCV all exhibited similar immunogenicity in a mouse model, inducing anti-JEV and anti-EDII 101/106/107 epitope-specific antibodies. However, the EDIII 329/331/389 epitope-specific IgG antibody and neutralizing antibody titers were significantly lower for FICV-immunized and FIV-immunized mouse serum than for UCV-immunized. Formalin inactivation seems to alter the antigenic structure of the E protein, which may reduce the potency of commercially available JEV vaccines. Virus inactivation by H2O2, but not by UV or by short-duration and higher temperature formalin treatment, is able to maintain the antigenic structure of the JEV E protein. Thus, an alternative inactivation method, such as H2O2, which is able to maintain the integrity of the E protein may be essential to improving the potency of inactivated JEV vaccines.

Comparison of β-Propiolactone and Formalin Inactivation on Antigenicity and Immune Response of West Nile Virus.

West Nile Virus (WNV) is a pathogenic arbovirus that belongs to genus Flavivirus under family Flaviviridae. Till now there are no approved vaccines against WNV for human use. In this study, the effect of two alkylating agents, formaldehyde and β-PL, generally used for inactivated vaccine preparation, was assessed on the basis of antigenic and immunogenic potential of the inactivated WNV. Lineage 5 WNV isolates were inactivated by both formalin and β-PL treatments. Inactivation was confirmed by repeated passage in BHK-21 cell line and infant mice. Viruses inactivated by both the treatments showed higher antigenicity. Immune response in mice model showed serum anti-WNV antibody titre was moderately higher in formalin inactivated antigen compared to β-PL inactivated antigen. However, no significant differences were observed in neutralization antibody titre. In conclusion, we can state that both formaldehyde and β-PL inactivation processes were found to be equally efficient for inactivation of WNV. However, they need to be compared with other inactivating agents along with study on cell mediated immune response.

Efficacy of single dose of an inactivated porcine circovirus type 2 (PCV2) whole-virus vaccine with oil adjuvant in piglets

BackgroundPost-weaning multisystemic wasting syndrome (PMWS) associated with PCV2 is one of the most costly diseases currently faced by the swine industry. The development of effective vaccines against PCV2 infection has been accepted as an important strategy in the prophylaxis of PMWS.MethodsIn the present study, a PK-15 cell-adapted formalin-inactivated prototype vaccine candidate was prepared using a strain of PCV2 from China. Inactivation of the virus was accomplished using a standard formalin inactivation protocol. The protective properties of the inactivated PCV2 vaccine were evaluated in piglets. Ten 28-day-old pigs were randomly assigned to two groups, each with five. Group 1 was vaccinated intramuscularly with the inactivated virus preparation; Group 2 received sterile PBS as a placebo. By 28 days post-vaccination (DPV), Groups 1 and 2 were challenged intranasally and intramuscularly with 5 × 107 TCID50 of a virulent PCV2 isolate.ResultsThe vaccinated pigs seroconverted to PCV2 and had high levels of serum antibodies to PCV2 at 28 days after vaccination, whereas the control pigs remained seronegative. No significant signs of clinical disease were recorded following the challenge with PCV2, but moderate amounts of PCV2 antigen were detected in most lymphoid organs of the control pigs. PCV2 was detected in two out of the five vaccinated pigs. Furthermore, pathological lesions and viremia were milder in the vaccinated group.ConclusionsThe obtained results indicate that the inactivated PCV2 virus vaccine with an oil adjuvant induce an immunological response in pigs that appears to provide protection from infection with PCV2. The vaccine, therefore, may have the potential to serve as a vaccine aimed to protect pigs from developing PMWS.

Assessment of immunogenic potential of Vero adapted formalin inactivated vaccine derived from novel ECSA genotype of Chikungunya virus

The recent resurgence of Chikungunya virus (CHIKV) in India and Indian Ocean Islands with unusual clinical severity is a matter of great public health concern. Despite the fact that CHIKV resurgence is associated with epidemic of unprecedented magnitude, no approved licensed vaccine is currently available. In the present study, a Vero cell adapted purified formalin inactivated prototype vaccine candidate was prepared using a current Indian strain implicated with the explosive epidemic during 2006. The bulk preparation of the vaccine candidate was undertaken in microcarrier based spinner culture using cytodex-1 in virus production serum free medium. The inactivation of the virus was accomplished through standard formalin inactivation protocol. The mice were immunized subcutaneously with alhydrogel gel formulation of inactivated virus preparation. The assessment of both humoral and cell-mediated immune response was accomplished through ELISA, plaque reduction neutralization test (PRNT), microcytotoxicity assay and cytokine production assay. The results revealed that formalin inactivated vaccine candidate induced both high titered ELISA (1:51,200) and plaque reduction neutralizing antibodies (1:6400) with peak antibody titer being observed during 6–8 weeks of post-vaccination. In the absence of suitable murine challenge model, the protective efficacy was established by both in vitro and in vivo neutralization tests. Further assessment of cellular immunity through in vitro stimulation of spleenocytes from immunized mice revealed augmentation of high levels of both pro- and anti-inflammatory cytokines, indicating a mixed balance of Th1 and Th2 response. These findings suggest that the formalin inactivated Chikungunya vaccine candidate reported in this study has very good immunogenic potential to neutralize the virus infectivity by augmenting both humoral and cell-mediated immune response.