Human Interferon-alpha Research Articles

A De Novo Strategy To Improve Pharmacokinetics of Proteins from mRNA Therapeutics via Zwitterionic Polypeptide Fusion.

Achieving therapeutic efficacy in protein replacement therapies requires sustaining pharmacokinetic (PK) profiles, while maintaining the bioactivity of circulating proteins. This is often achieved via PEGylation in protein-based therapies, but it remains challenging for proteins produced in vivo in mRNA-based therapies due to the lack of a suitable post-translational modification method. To address this issue, we integrated a genetically encoded zwitterionic polypeptide, EKP, into mRNA constructs to enhance the PK properties of product proteins. Composed of alternating glutamic acid (E), lysine (K), and proline (P), EKP exhibits unique superhydrophilic properties and low immunogenicity. Our results demonstrate that EKP fusion significantly extends the circulation half-life of proteins expressed from mRNA while preserving their bioactivity using human interferon alpha and Neoleukin-2/15 as examples. This EKP fusion technology offers a new approach to overcoming the current limitations in mRNA therapeutics and has the potential to significantly advance the development of mRNA-based protein replacement therapy.

Production of Recombinant Human Interferon alpha 2b (rhIFN?-2b) in genetically engineered strain of Bacillus subtilis

Interferon alpha 2b (IFN?-2b) is a critical therapeutic protein with immunomodulatory properties, widely used in the treatment of various diseases, including viral infections and cancer. This study presents a comprehensive methodology for the cloning and characterization of the IFN?-2b gene, including the identification of point mutations, followed by expression in Bacillus subtilis. The research begins with gene isolation, primer design, RNA extraction, cDNA synthesis, and PCR amplification. Cloning into a T/A vector and subsequent sequencing reveal point mutations within the gene. The transformed construct is expressed in Bacillus subtilis, and the resulting protein is analyzed using SDS-PAGE. The identification of point mutations and successful protein expression in Bacillus subtilis. This study provides valuable insights into the IFN?-2b gene and its potential applications.

Use of Recombinant Human Interferon Alpha in felines infected with FeLV

Abstract. The feline leukemia virus (FeLV) is a retrovirus that belongs to the genus Gammaretrovirus and exclusively affects felids. The virus carries the reverse transcriptase enzyme, which integrates into the host's DNA permanently, leading to different clinical conditions. The main complications caused by FeLV are hematopoietic neoplasms, immune-mediated disorders, neurological alterations and immunosuppression, among other ailments. The disease has no effective treatment, but antiviral and immunomodulatory drugs are still under trial. Using Recombinant Human Interferon Alpha has shown to be helpful in reducing clinical symptoms and controlling opportunistic infections, increasing the quality and life expectancy of cats infected with FeLV. Retroviruses are among the main causes of death in domestic cats. The lack of knowledge about the disease among guardians and growing cat population in Brazil are helping to increase the number of FeLV cases. Although the disease lacks a universal veterinary medical protocol, there are new studies and information available that can help to treat infected animals, improving their quality of life and expectancy and, in some cases, potentially eliminating the virus. This fact clashes with another reality: outdated professionals who are not familiar with the guidelines for Feline Retrovirus Testing and Management from the AAFP (American Association of Feline Practitioners), an international entity that since 1971 has been working to improve feline health by providing evidence-based guidelines for veterinary practices. Population growth and diseases that exclusively affect the species also highlight the need to include a deeper understanding of feline health in the curricula of undergraduate courses, preparing these future professionals to meet the demand for updated veterinarians with sufficient knowledge to care for the species.

Construction of viral-based expression vectors for high-level production of human interferon alpha 2b in plants

Human interferon (hINF) alpha 2b is clinically important pharmaceutical product included in combinatory therapy against chronic hepatitis C and B and complex therapy against several cancer diseases. Here, we created the genetic constructions, based on genome elements of potato virus X (PVX), carrying the infα2b gene for transient expression in plant cells. The created plasmid vector constructions were tested through Agrobacterium-mediated transient gene expression method in two plant species—Nicotiana benthamiana and Ocimum basilicum (sweet basil). Production of recombinant hINF alpha 2b was more efficient in N. benthamiana than that in O. basilicum plants. The average yield of hINF alpha 2b produced in N. benthamiana plants was 0.56 mg/g of fresh leaf weight (FW) or 6% of the total soluble cell proteins (TSP). The maximal level reached up to 1.2 mg/g FW or 9% TSP. We estimated that about 0.67 mg of hINF can be obtained from one N. benthamiana plant. The yield of hINF alpha 2b obtained with the PVX-based expression cassette was about 80 times higher than the yield of hINF alpha 2b obtained with a simple expression cassette in which the infα2b gene was controlled by the 35S promoter of cauliflower mosaic virus.Key points• PVX-based expression vectors provide efficient transient expression of infα2b gene• N. benthamiana plants can produce human interferon alpha 2b at high levels• The yield of the hINF α2b reached up to 1.2 mg/g of fresh leaf weight

An extracellular humanized IFNAR immunocompetent mouse model for analyses of human interferon alpha and subtypes

ABSTRACT Type I interferons (IFN-Is) have key roles in immune defense and treatments for various diseases, including chronic hepatitis B virus (HBV) infection. All IFN-Is signal through a shared IFN-I heterodimeric receptor complex comprising IFN-α receptor 1 (IFNAR1) and IFNAR2 subunits, but differences in antiviral and immunomodulatory responses among IFN-I subtypes remain largely unknown. Because the IFN-IFNAR interactions are species-specific, mice exhibit weak responses to human IFN-I. To more fully characterize the actions of human IFN-α and its subtypes in vivo, a gene targeting strategy was employed to generate gene knock-in mice with extracellular-humanized IFNAR1/2 (IFNAR-hEC) in the C57BL/6N strain. IFNAR-hEC mice actively responded to human IFN-I, and endogenous mouse IFN-I signalling remained active in heterozygous mice (Ifnar hEC/+). Analyses of IFNAR-hEC mice and isolated cells showed that human IFN-α2 and α14 subtypes exerted differential effect on the activation of JAK-STAT signalling and immune responses. Compared with IFN-α2, IFN-α14 induced greater activation of STAT1/2 and IFN-stimulated genes, synergistically elicited IFN-α and -γ signalling, and induced higher numbers of antigen-specific CD8+ T cells. Moreover, IFNAR-hEC mice with HBV replication displayed long-term viral suppression upon treatment with the clinically-used PEGylated hIFN-α2. These results indicate that IFNAR-hEC mice may be useful for elucidating antiviral and immunomodulatory functions of human IFN-Is and for conducting preclinical studies. A better understanding of the distinct activities of IFN-α subtypes can provide insights concerning the development of improved IFN-based therapy.

Detection of interferon alpha and beta receptor subunit 1 (IFNAR1) loss-of-function Glu386∗ variant by tri-allelic genotyping

Mutations of the human interferon alpha and beta receptor subunit 1 (IFNAR1) gene are associated with severe viral infections. Individuals homozygous for the Glu386∗ variant have impaired type I interferon signalling and can suffer severe illness when exposed to certain viruses and live attenuated virus vaccines. Glu386∗ heterozygotes are clinically unaffected, but can pass the variant allele to their descendants. We aimed to develop an assay that can identify IFNAR1 Glu386∗ homozygotes and heterozygotes to support urgent clinical diagnosis, and that can use dried blood spots (DBS) sent at ambient temperature to overcome geographical logistical challenges in the South Pacific region. The tri-allelic genotyping assay interrogates a single nucleotide polymorphism (rs201609461) located in IFNAR1. The reference allele G encodes for wild-type IFNAR1. Minor alleles A (c.1156G>A) and T (c.1156G>T) encode for Glu386Lys and a truncated IFNAR1 protein (p.Glu386∗), respectively. Synthetic oligonucleotides were mixed in equal molar ratio to create six different genotypes which were randomly assigned to 960 genotyping reactions by R software. Three different fluorescence probes were designed to discriminate the three alleles (G, T and A) within a pair of flanking primers in a single genotyping reaction. The assay discriminated all three alleles using DBS from Guthrie cards randomly spiked with synthetic oligonucleotides. We correctly identified all the different genotypes in 960 reactions in these blinded experiments. These findings validate the genotyping assay for rapidly identifying the IFNAR1 Glu386∗ variant from DBS.

Expression and purification of human interferon alpha 2a (IFNα2a) in the methylotrophic yeast Pichia pastoris

Human interferon alpha 2a (IFNα2a) is a secreted glycoprotein that exerts a wide spectrum of biological effects, such as triggering of antiviral, antitumor and immunosuppressive responses. IFNα2a is used as pharmaceutical polypeptide in chronic hepatitis C virus (HCV) infection, chronic myelogenous leukemia, advanced renal cell carcinoma, and metastatic malignant melanoma. So far, the pharmaceutical polypeptide of this cytokine is produced in prokaryotic expression systems (E. coli). Here we report the expression and purification of recombinant human IFNα2a in the methylotrophic yeast Pichia pastoris. The cDNA encoding for human IFNα2a, modified to bear the P. pastoris codon bias, was cloned into the pPinkα-HC vector in order to be expressed as a secreted protein upon induction. Proper expression and secretion of recombinant human IFNα2a (approximately 19 kDa) was confirmed by PCR-sequencing, SDS-PAGE and Western blot analysis following methanol-induced expression in a number of individual transformed strains. Purification of the recombinant protein was performed by affinity chromatography, achieving a robust yield of purified active form. The purified recombinant protein showed an impressive stability to thermal denaturation as observed by Differential Scanning Fluorimetry. The biological activity of the P. pastoris-produced IFNα2a was confirmed in A549 and HT29 cells by monitoring transcriptional up-regulation of a panel of known interferon-stimulated genes (ISGs). Our results document that the P. pastoris expression system is a suitable system for producing biologically functional IFNα2a in a secreted form.

Specific sequence mutations in a long-lasting rhIFN-α2b version reduce in vitro and in vivo immunogenicity and increase in vitro protein stability

For decades, recombinant human interferon alpha (rhIFN-α2b) has been used to treat emerging and chronic viral diseases. However, rhIFN-α2b is immunogenic and has a short in vivo half-life. To solve these limitations, two long-lasting hyperglycosylated proteins with reduced immunogenicity were developed and designated as 4N-IFN(VAR1) and 4N-IFN(VAR3).Here, we continue to study the relevant characteristics of these therapeutic candidates. Thus, we demonstrated that both de-immunized IFN versions elicited significantly lower neutralizing antibody responses than the original molecule in HLA-DR1 transgenic mice, confirming our previous in vitro protein immunogenicity data. Also, we found that these biobetters exhibited remarkable stability when exposed to different physical factors that the protein product may encounter during its production process and storage, such as low pH, thermal stress, and repeated freezing/thawing cycles. Taking into consideration our previous and present results, 4N-IFN(VAR1) and 4N-IFN-4N(VAR3) appear to be valuable candidates for the treatment of human viral diseases.

Deployment of metabolic heat rate based soft sensor for estimation and control of specific growth rate in glycoengineered Pichia pastoris for human interferon alpha 2b production

Lack of appropriate process models, reliable online sensors, and process variability in bioprocess systems are poising challenges in real-time monitoring and control of critical process parameters (CPPs). This present investigation deals with the development of a non-invasive soft sensor by utilizing metabolic heat rate as input signal for online estimation of specific growth rate (μest) during the induction phase of glycoengineered Pichia pastoris for human interferon-alpha 2b (huIFNα2b) production. Feedforward strategy employing a predetermined exponential feeding of methanol during the induction phase was dealt at defined setpoint values (μSP). Standard PID controller with predetermined gain values regulated methanol feeding in accordance with the deviation from the μSP value. An adaptive PID (gain scheduling) significantly minimized the deviation of μ from its μSP value, reduced the amplitude of oscillation and achieved long-term controller stability. Robust control of methanol feeding by adaptive PID resulted in a 1.5 and 2.2-fold increase in productivity of huIFNα2b compared to standard PID and feedforward controls respectively. Moreover, adaptive PID control facilitated narrow range control of μ for longer durations (> 20 h) with a low average tracking error (< 6%) enumerating its scope of application in therapeutic protein production in near future.

Anti-Viral Activities of Umbilical Cord Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Against Human Respiratory Viruses

The endemic and pandemic caused by respiratory virus infection are a major cause of mortality and morbidity globally. Thus, broadly effective antiviral drugs are needed to treat respiratory viral diseases. Small extracellular vesicles derived from human umbilical cord mesenchymal stem cells (U-exo) have recently gained attention as a cell-free therapeutic strategy due to their potential for safety and efficacy. Anti-viral activities of U-exo to countermeasure respiratory virus-associated diseases are currently unknown. Here, we tested the antiviral activities of U-exo following influenza A/B virus (IFV) and human seasonal coronavirus (HCoV) infections in vitro. Cells were subject to IFV or HCoV infection followed by U-exo treatment. U-exo treatment significantly reduced IFV or HCoV replication and combined treatment with recombinant human interferon-alpha protein (IFN-α) exerted synergistically enhanced antiviral effects against IFV or HCoV. Interestingly, microRNA (miR)-125b, which is one of the most abundantly expressed small RNAs in U-exo, was found to suppress IFV replication possibly via the induction of IFN-stimulated genes (ISGs). Furthermore, U-exo markedly enhanced RNA virus-triggered IFN signaling and ISGs production. Similarly, human nasal epithelial cells cultured at the air-liquid interface (ALI) studies broadly effective anti-viral and anti-inflammatory activities of U-exo against IFV and HCoV, suggesting the potential role of U-exo as a promising intervention for respiratory virus-associated diseases.

The Enhancing Effects of 10% PBS Washout of Holocene Minerals on HuIFN-αN3 Inducing Capacity of NDV ZG1999HDS or Sendai virus (Cantell Strain).

Different strains of Newcastle disease viruses (NDV) or Sendai viruses (SV) are used to induce the production of human leukocyte multi subtype interferon-alpha (HuIFN-αN3). Their inducing capacity can be enhanced in different ways. One includes 10% PBS washout of Holocene minerals (HM). The presented study aims to compare the HuIFN-αN3 inducing capacity of NDV ZG1999HDS or SV (Cantell strain) strain in vitro, and to evaluate the enhancing effect of 10% PBS washouts of HM on both viruses. The NDV strains’ ZG1999HDS interferon inducing capacity (483.23 ± 4.5 pg/mL) was similar to that of the SV (Cantell strain) (584.16 ± 5.9 pg/mL). It was shown that the HuIFN-αN3 inducing capacity of the strain of NDV ZG1999HDS can be strongly enhanced with 10% PBS washout of HM to 3818.21 ± 41.9 pg/mL and 4790.34 ± 33.5 pg/mL with SV (Cantell strain), u. The RP-HPLC analyses of such HuIFN-αN3 induced with the strain of NDV ZG1999HDS show the difference to SV (Cantell strain) induced HuIFN-αN3 in the absence of subtype α14 and the lower level of the subtype α1. The possible ways of such enhancement were also studied and it was postulated that the Fe2+ ions from 10% PBS washouts of HM, while stimulating the reactive oxygen species (ROS) and nitric oxide (NO) formation, activate the transcription factor NF- κB and consequently the production of HuIFN-αN3.

Use Intein Cleavable Polyhydroxyalkanoate Synthase Fusions to Improve Protein Solubility.

Recombinant E. coli producing intein-cleavable polyhydroxyalkanoate synthase fusions mediates the intracellular formation of polyhydroxyalkanoate (PHA) particles densely coated with intein-cleavable target protein fusion. These PHA particles can be efficiently purified from lysed cells. The self-cleaving intein performs as a bio-linker between the PHA synthase and the target protein. The tagless target protein can be released as pure soluble protein from the PHA particles by a simple pH reduction to 6.0. Here we describe that PHA particles serve as bioseparation resin for purification of soluble target proteins with pharmaceutical grade purity, similar to commercial affinity separation technologies. This cost-effective technique does not involve multiple complicated protein purification procedures, and we have exploited this approach to purify six target proteins: green fluorescent protein (GFP) from A. victoria, antigen Rv1626 from M. tuberculosis, the immunoglobulin G (IgG) binding ZZ domain of protein A derived from Staphylococcus aureus, human tumor necrosis factor alpha (TNFα), human granulocyte colony-stimulating factor (G-CSF), and human interferon alpha 2b (IFNα2b).

Development of highly stable and de-immunized versions of recombinant alpha interferon: Promising candidates for the treatment of chronic and emerging viral diseases

Human interferon alpha (hIFN-α) administration constitutes the current FDA approved therapy for chronic Hepatitis B and C virus infections. Additionally, hIFN-α treatment efficacy was recently demonstrated in patients with COVID-19. Thus, hIFN-α constitutes a therapeutic alternative for those countries where vaccination is inaccessible and for people who did not respond effectively to vaccination. However, hIFN-α2b exhibits a short plasma half-life resulting in the occurrence of severe side effects. To optimize the cytokine's pharmacokinetic profile, we developed a hyperglycosylated IFN, referred to as GMOP-IFN.Given the significant number of reports showing neutralizing antibodies (NAb) formation after hIFN-α administration, here we applied the DeFT (De-immunization of Functional Therapeutics) approach to develop functional, de-immunized versions of GMOP-IFN. Two GMOP-IFN variants exhibited significantly reduced ex vivo immunogenicity and null antiproliferative activity, while preserving antiviral function. The results obtained in this work indicate that the new de-immunized GMOP-IFN variants constitute promising candidates for antiviral therapy.

Shared and Unique Features of Human Interferon-Beta and Interferon-Alpha Subtypes.

Type I interferons (IFN-I) were first discovered as an antiviral factor by Isaacs and Lindenmann in 1957, but they are now known to also modulate innate and adaptive immunity and suppress proliferation of cancer cells. While much has been revealed about IFN-I, it remains a mystery as to why there are 16 different IFN-I gene products, including IFNβ, IFNω, and 12 subtypes of IFNα. Here, we discuss shared and unique aspects of these IFN-I in the context of their evolution, expression patterns, and signaling through their shared heterodimeric receptor. We propose that rather than investigating responses to individual IFN-I, these contexts can serve as an alternative approach toward investigating roles for IFNα subtypes. Finally, we review uses of IFNα and IFNβ as therapeutic agents to suppress chronic viral infections or to treat multiple sclerosis.

Pharmacokinetics Versus In Vitro Antiproliferative Potency to Design a Novel Hyperglycosylated hIFN-α2 Biobetter.

IFN4N is a glycoengineered version of recombinant human interferon alpha 2 (rhIFN-α2) that was modified to exhibit four N-glycosylation sites. It shows reduced in vitro specific biological activity (SBA) mainly due to R23 mutation by N23. However, it has improved pharmacokinetics and led to a high in vivo antitumor activity in mice. In order to prepare a new IFN-based biobetter, this work compares the influence of glycosylation (affecting pharmacokinetics) with the in vitro antiproliferative SBA on the in vivo efficacy. Based on IFN4N, three groups of muteins were designed, produced, and characterized. Group A: variants with the same glycosylation degree (4N) but higher in vitro antiproliferative SBA (R23 restored); group B: muteins with higher glycosylation degree (5N) but similar in vitro antiproliferative activity; and group C: variants with improved glycosylation (5N and 6N) and in vitro antiproliferative bioactivity. Glycoengineering was successful for improving pharmacokinetics, and R23 restoration considerably increased in vitro antiproliferative activity of new muteins compared to IFN4N. Hyperglycosylation was able to improve the in vivo efficacy similarly to or even better than R23 restoration. Additionally, the highest glycosylated mutein exhibited the lowest immunogenicity. Hyperglycosylation constitutes a successful strategy to prepare a novel IFN biobetter.

The Effect of Recombinant Human Interferon Alpha Nasal Drops to Prevent COVID-19 Pneumonia for Medical Staff in an Epidemic Area

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Use of immunostimulants in the successful treatment of a clinical EEHV1A infection in an Asian elephant (Elephas maximus)

Elephant endotheliotropic herpesvirus haemorrhagic disease (EEHV‐HD) poses a significant threat to the captive population of juvenile Asian elephants (Elephas maximus) and also affects elephants in the wild. In human and veterinary medicine, increasing attention is returning to the interferon system, a crucial mediator in the immune response for control of infection. We describe the first reported use of the Zelnate DNA immunostimulant and recombinant human interferon alpha (rhIFNα) as additional medications in the successful treatment of a case of EEHV1A‐HD in a juvenile Asian elephant at a UK zoo. Despite an exponential rise in viraemia to a peak of 2.82×106 viral genomic equivalents/ml, only mild clinical signs developed and the calf survived with no adverse effects attributed to the novel treatments. This case is compared with a previous fatal case within the same herd where Zelnate and rhIFNα were not given.

Clinical characteristics and predictors of the duration of SARS-CoV-2 viral shedding in 140 healthcare workers.

BackgroundEpidemiological and clinical features of patients with COVID‐19 have been reported, but none of them focused on medical staff, and few predictors of the duration of viral shedding have been reported. It is urgent to help healthcare workers prevent and recover quickly from the coronavirus disease 2019 (COVID‐19).MethodsWe enrolled 140 medical workers with COVID‐19 in Wuhan. Epidemiological, demographic, clinical, laboratory, radiological treatment and clinical outcome data were collected, and predictors of the duration of viral shedding were explored through multivariable linear regression analysis.ResultsThe medical staff with COVID‐19 presented mild clinical symptoms and showed a low frequency of abnormal laboratory indicators. All the medical staff were cured and discharged, of whom 96 (68.6%) were female, 39 (27.9%) had underlying diseases, the median age was 36.0 years, and 104 (74.3%) were infected whilst working in hospital. The median duration of viral shedding was 25.0 days (IQR:20.0–30.0). Multivariable linear regression analysis showed reducing viral shedding duration was associated with receiving recombinant human interferon alpha (rIFN‐α) treatment, whilst the prolonged duration of viral shedding correlated with the use of glucocorticoid treatment, the durations from the first symptom to hospital admission and the improvement in chest computed tomography (CT) evidence. Moreover, infected healthcare workers with lymphocytes less than 1.1 × 109/L on admission had prolonged viral shedding.ConclusionMedical staff with timely medical interventions show milder clinical features. Glucocorticoid treatment and lymphocytes less than 1.1 × 109/L are associated with prolonged viral shedding. Early admission and rIFN‐α treatment help shorten the duration of viral shedding.

Clinical and Hematological Follow-Up of Long-Term Oral Therapy with Type-I Interferon in Cats Naturally Infected with Feline Leukemia Virus or Feline Immunodeficiency Virus.

Simple SummaryThe viruses which produce feline leukemia (FeLV) and feline immunodeficiency (FIV) attack cells involved in the immune response. As specific drugs are either nonexistent, produce secondary effects, or are expensive, a therapeutic possibility is using nonspecific immunostimulants, such as human interferon alpha. We used this drug to treat 27 cats infected by FeLV and 31 infected by FIV. All cats were naturally infected and treatment was administered orally by their owners for four months. Participating cats were evaluated in our clinics at mid-treatment (M2), end of treatment (M4), and 4–8 months after the end of treatment (M10). We observed that treatment was well tolerated by the cats (as it did not affect the liver or the kidney functions), and improved most of the parameters analyzed (clinic, anemia, white cell counts, and CD4+/CD8+ ratio) as long as it was administered. However, 4–8 months after it was discontinued, though most animals remained clinically healthy, many of these parameters had rebounded to initial values or values even worse than the initial values. Thus, more studies should be conducted with longer administration of this drug to evaluate tolerability and sustained improvement of diseases produced by these two viruses which may lead to death.Feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV), two of the most important pathogens of cats, produce chronic systemic diseases with progressive death of cells involved in the immune response, ultimately leading to death. Immunostimulants is one of the few alternatives to the symptomatic treatment. In this study, 27 naturally FeLV-infected (FeLV+) and 31 naturally FIV-infected (FIV+) cats were administered orally by their owners 60 IU/day of recombinant human interferon alpha (rHuIFN-α) for four months in alternate weeks. Clinical status was evaluated and blood samples collected at four different visits or months (M): pretreatment (M0), mid-treatment (M2), end of treatment (M4), and 4–8 months after end of treatment (M10). Most cats ostensibly improved their clinical status, and many became asymptomatic. rHuIFN-α treatment improved the anemic processes observed at M0 (at least in cats with mild or moderate anemia) and leukocyte counts, including a more favorable CD4+/CD8+ ratio. An increase in the serum gammaglobulin concentration was seen in 80% of the cats. Despite observing an obvious favorable progress in the clinical, biopathological, and CD4+/CD8+ values during treatment, almost invariably all the parameters analyzed worsened after treatment discontinuation (M10), which suggests that the interferon-α protocol should be either extended or include additional cycles for a long-lasting benefit in FeLV+ and FIV+ cats.

Effect of biphasic temperature regime on therapeutic recombinant protein production in the green alga Chlamydomonas reinhardtii

Microalgae are increasingly being considered for recombinant protein production because of low cultivation costs, absence of endotoxins and insusceptibility to human infectious agents. Despite these advantages, the yield of recombinant protein produced in microalgae is still low compared to more established expression systems and optimization at the genetic and cultivation levels is required for this new system to be economically viable. This study investigates the effect of biphasic temperature regimes on the yield of recombinant human interferon alpha 2a (IFN-α2a), a therapeutic protein known for its anti-cancer and anti-viral properties, produced by the model green alga Chlamydomonas reinhardtii (Cr.IFN-α2a). Biphasic growth is commonly employed to increase recombinant protein production in mammalian cell lines used for commercial production of therapeutic proteins, with a lowering of the temperature resulting in higher yields. In this study, lowering the temperature from 25 °C to 15 °C in mid-exponential growth phase increased the accumulation of Cr.IFN-α2a by 3.3-fold while it slowed down the growth of the three C. reinhardtii transgenic lines tested. In contrast, a rise of temperature from 25 °C to 35 °C accelerated cell growth, while negatively impacting the production of Cr.IFN-α2a. After a two-step chromatography purification, the Cr.IFN-α2a produced was estimated to be 53% pure with a yield of 90 μg/L of culture. The amino acid sequence of Cr.IFN-α2a was confirmed by mass spectrometry. However, the anti-viral activity of Cr.IFN-α2a was found to be 10 times lower than the human IFN-α2a standard produced using E. coli when challenged in a cytopathic effect (CPE) assay, likely due to the formation of aggregates. While the molecular mechanisms driving the accumulation of Cr.IFN-α2a at lower temperature remains unclear, our results support that reducing the temperature at the peak of expression is a valid strategy to increase the yield of recombinant Cr.IFN-α2a in C. reinhardtii.