Camelid VHHs Fused to Human Fc Fragments Provide Long Term Protection Against Botulinum Neurotoxin A in Mice

The bacterium Clostridium botulinum is the causative agent of botulism—a severe intoxication caused by botulinum neurotoxin (BoNT) and characterized by damage to the nervous system. In an effort to develop novel C. botulinum immunotherapeutics, camelid single-domain antibodies (sdAbs, VHHs, or nanobodies) could be used due to their unique structure and characteristics. In this study, VHHs were produced using phage display technology. A total of 15 different monoclonal VHHs were selected based on their comlementarity-determining region 3 (CDR3) sequences. Different toxin lethal dose (LD50) challenges with each selected phage clone were conducted in vivo to check their neutralizing potency. We demonstrated that modification of neutralizing VHHs with a human immunoglobulin G (IgG)1 Fc (fragment crystallizable) fragment (fusionbody, VHH-Fc) significantly increased the circulation time in the blood (up to 14 days). At the same time, VHH-Fc showed the protective activity 1000 times higher than monomeric form when challenged with 5 LD50. Moreover, VHH-Fcs remained protective even 14 days after antibody administration. These results indicate that this VHH-Fc could be used as an effective long term antitoxin protection against botulinum type A

Genetic Passive Immunization with Adenoviral Vector Expressing Chimeric Nanobody-Fc Molecules as Therapy for Genital Infection Caused by Mycoplasma hominis.

Developing pathogen-specific recombinant antibody fragments (especially nanobodies) is a very promising strategy for the treatment of infectious disease. Nanobodies have great potential for gene therapy application due to their single-gene nature. Historically, Mycoplasma hominis has not been considered pathogenic bacteria due to the lack of acute infection and partially due to multiple studies demonstrating high frequency of isolation of M. hominis samples from asymptomatic patients. However, recent studies on the role of latent M. hominis infection in oncologic transformation, especially prostate cancer, and reports that M. hominis infects Trichomonas and confers antibiotic resistance to Trichomonas, have generated new interest in this field. In the present study we have generated specific nanobody against M. hominis (aMh), for which the identified target is the ABC-transporter substrate-binding protein. aMh exhibits specific antibacterial action against M. hominis. In an attempt to improve the therapeutic properties, we have developed the adenoviral vector-based gene therapy approach for passive immunization with nanobodies against M. hominis. For better penetration into the mucous layer of the genital tract, we fused aMh with the Fc-fragment of IgG. Application of this comprehensive approach with a single systemic administration of recombinant adenovirus expressing aMh-Fc demonstrated both prophylactic and therapeutic effects in a mouse model of genital M. hominis infection

Cross-Reactive Fc-Fused Single-Domain Antibodies to Hemagglutinin Stem Region Protect Mice from Group 1 Influenza a Virus Infection

The continued evolution of influenza viruses reduces the effectiveness of vaccination and antiviral drugs. The identification of novel and universal agents for influenza prophylaxis and treatment is an urgent need. We have previously described two potent single-domain antibodies (VHH), G2.3 and H1.2, which bind to the stem domain of hemagglutinin and efficiently neutralize H1N1 and H5N2 influenza viruses in vivo. In this study, we modified these VHHs with Fc-fragment to enhance their antiviral activity. Reformatting of G2.3 into bivalent Fc-fusion molecule increased its in vitro neutralizing activity against H1N1 and H2N3 viruses up to 80-fold and, moreover, resulted in obtaining the ability to neutralize H5N2 and H9N2 subtypes. We demonstrated that a dose as low as 0.6 mg/kg of G2.3-Fc or H1.2-Fc administered systemically or locally before infection could protect mice from lethal challenges with both H1N1 and H5N2 viruses. Furthermore, G2.3-Fc reduced the lung viral load to an undetectable level. Both VHH-Fc antibodies showed in vivo therapeutic efficacy when delivered via systemic or local route. The findings support G2.3-Fc as a potential therapeutic agent for both prophylaxis and therapy of Group 1 influenza A infection

Hypothetical mechanism of blocking nutrient acquisition through ABC transporter with aMh-FcG2a.

<p>Hypothetical mechanism of blocking nutrient acquisition through ABC transporter with aMh-FcG2a.</p

Determination of affinity constants.

<p>Binding of antigen to aMh-FcG2a was determined by surface plasmon resonance using Biacore 3000 (GE Healthcare). Antigen concentration series 5.95 nM; 11.9 nM; 59.5 nM and 595 nM (colour) and 1:1 fitting (black) interaction antigen to aMh-Fc. The fitted constant are k_a = 1.78⁴ M^-1s^-1 and k_d = 1.06⁻⁴ s^-1 which results <i>K</i>_<i>D</i> = 5.94*10⁻⁹ M (R_max = 428 RU; chi² = 11.8). Evaluation included double reference subtraction.</p

Titers of <i>M</i>. <i>hominis</i> in vaginal washes with “prophylactic” scheme of rAds inoculation.

<p>Vaginal washes were collected 5 days after <i>M</i>. <i>hominis</i> inoculation (6 days after rAds inoculation). Amount of <i>M</i>. <i>hominis</i> was evaluated with real-time PCR. The rAd5-CMV-PLAP-aMh-FcG2a group exhibited a significantly lower <i>M</i>. <i>hominis</i> titer (Student's t-test = 3.5; p<0.01). Ad-null n = 5, PBS n = 15, rAd5-CMV-PLAP-aMh-FcG2a n = 10, rAd5-CMV-PLAP-aMh-ILZ-HA n = 11. In the “prophylactic” scheme of rAds inoculation, the <i>M</i>. <i>hominis</i> titer was significantly decreased in the rAd5-CMV-PLAP-aMh-FcG2a group. The prophylactic scheme, however, is not practical for treating mycoplasma infection. In the “therapeutic” scheme, the number of animals diagnosed as positive at 7 days after inoculation with rAd5-CMV-PLAP-aMh-FcG2a (12 days after <i>M</i>. <i>hominis</i> inoculation) was significantly lower than that of the other groups. Nevertheless the mycoplasma titer was not significantly different among the infected animals in any of the groups and not informative due to the large variability in counts and the low number of infected animals in the rAd5-CMV-PLAP-aMh-FcG2a group (n = 2 at 3 days and n = 1 at 7 days).</p

rAd5-CMV-PLAP-aMh-FcG2a and control rAd5-CMV-PLAP-aMh-ILZ-HA constructions and purification.

<p>(A) Scheme of expression cassettes for rAd5-CMV constructions; (B) sizing of purified Ad5-CMV-PLAP-aMh-FcG2a corresponding to virions suspensions with a particle size of ~100 nM.</p

Dimer formations of aMh-FcG2a.

<p>Protein samples aMh-FcG2a were prepared before electrophoresis in sample buffer with or without DTT (as reducing agent), canonical mouse IgG2a was prepared with DTT and used as control. Protein bands ~42 and ~84 kDa corresponding monomer and dimer forms of aMh-Fc; protein bands ~54 kDa and 25 kDa corresponding heavy and light chains of canonical mouse IgG2a.</p