Dosing Considerations for Antibodies Against COVID-19.

At present, no cure is available for COVID-19 but vaccines, antiviral drugs, immunoglobulins, or the combination of immunoglobulins with antiviral drugs have been suggested and are in clinical trials. The purpose of this paper is to discuss the role of a pharmacokinetic and viral load analysis as a basis for adjusting immunoglobulin dosing to treat COVID-19. We reviewed the pre-clinical and clinical literature that describes the impact of a high antigen load on pharmacokinetic data following antibody treatment. Representative examples are provided to illustrate the effect of high viral and tumor loads on antibody clearance. We then highlight the implications of these factors for facilitating the development and dosing of hyperimmune anti-SARS CoV2 immunoglobulin. Both nonclinical and clinical examples indicate that high antigen loads, whether they be viral, bacterial, or tumoral in origin, result in increased clearance and decreased area under the curve and half-life of antibodies. A dosing strategy that matches the antigen load can be achieved by giving initially high doses and adjusting the frequency of dosing intervals based on pharmacokinetic parameters. We suggest that study design and dose selection for immunoglobulin products for the treatment of COVID-19 require special considerations such as viral load, antibody-virus interaction, and dosing adjustment based on the pharmacokinetics of the antibody.

Considerations for pharmacokinetic assessment of immunoglobulins: Gammagard in very low birth weight neonates with and without baseline-correction.

BACKGROUND: Immunoglobulins are widely used across multiple therapeutic areas such as immunodeficiency syndromes, infection and autoimmune diseases. The pharmacokinetics (PK) of immunoglobulins are well characterized in adults, but very little is known about the PK of immunoglobulins in neonates and infants. OBJECTIVE: The objective of the present study was to characterize the PK of Gammagard, an immunoglobulin, in very low birth weight preterm neonates. METHOD: Gammagard concentration-time data from very low birth weight neonates (bodyweight range 0.78-1.38 kg, n = 20) following intravenous administration of 500 mg/kg and 750 mg/kg were obtained from the literature. The data were analyzed with and without baseline correction using extensive blood samples (8 blood samples). Model-independent (non-compartmental) analysis was used to characterize the PK of Gammagard. RESULTS: Based on uncorrected baseline concentration-time data, the clearance and half-life of Gammagard were 3.1 ± 0.7 mL/day and 22 ± 6 days, respectively. Based on corrected baseline concentration-time data, the clearance and half-life of Gammagard were 20.2 ± 7.4 mL/day and 5.3 ± 2.2 days, respectively. CONCLUSION: The dose of immunoglobulins should be adjusted based on the PK of baseline corrected rather than baseline uncorrected profiles because baseline corrected PK parameters especially half-life reconciles with PK principles.

Fc-Fusion Drugs Have FcγR/C1q Binding and Signaling Properties That May Affect Their Immunogenicity.

Fusing the human immunoglobulin G1 (IgG1) constant region (Fc-domain) to therapeutic proteins or peptides increases their circulating plasma half-life via neonatal Fc receptor (FcRn) binding and recycling. However, Fc-mediated interactions with other molecules including complement C1q and Fc gamma receptors (FcγRs) can have immunological consequences and the potential to modulate the immunogenicity of Fc-fusion therapeutics. In a comparative study, we carried out a comprehensive assessment of Fc-mediated interactions for five FDA-approved Fc-fusion therapeutics. C1q binding and complement activation were measured by ELISA, while FcγR binding and signaling were evaluated using BW5147:FcγR-ζ reporter cell lines. We demonstrate that FIX-Fc and FVIII-Fc bound C1q as well as activating and inhibitory FcγRs (I, IIA, IIB, IIIA). These coagulation factor Fc-fusions also signaled via FcγRIIIA, and to a lesser extent via FcγRI and FcγRIIB. TNFR-Fc and CTLA4-Fc bound FcγRI, while TNFR-Fc also bound FcγRIIIA, but these interactions did not result in FcγR signaling. Our comprehensive assessment demonstrates that (i) different Fc-fusion drugs have distinct C1q/FcγR binding and signaling properties, (ii) FcγR binding does not predict signaling, and (iii) the fusion partner (effector molecule) can influence Fc-mediated interactions.

Brucella abortus bacA mutant induces greater pro-inflammatory cytokines than the wild-type parent strain.

The inner-membrane protein BacA affects Brucella LPS structure. A bacA deletion mutant of Brucella abortus, known as KL7 (bacA(mut)-KL7), is attenuated in BALB/c mice and protects against challenge. Thus, bacA mutation was a candidate for incorporation into live attenuated vaccines. We assessed bacA(mut)-KL7 in 2 additional mouse strains: the more resistant C57BL/6 that produces interferon-gamma throughout the infection and the highly susceptible interferon-gamma-deficient C57BL/6 in which brucellae exhibit continual exponential growth. While it was hypothesized that bacA(mut)-KL7 would exhibit even greater attenuation relative to its parent strain B. abortus 2308 in C57BL/6 mice than it did in BALB/c mice, this was not the case. Moreover, it was more pathogenic in C57BL/6 interferon-gamma-deficient mice than 2308 causing abscesses and wasting even though the splenic loads of bacA(mut)-KL7 were significantly lower. These 2 observations were correlated, respectively, with an ability of IFNgamma-activated macrophages to equivalently control strains 2308 and bacA(mut)-KL7 and the ability of bacA(mut)-KL7 organism and its LPS to induce greater amounts of pro-inflammatory cytokines than 2308. We conclude that attenuation properties of bacA mutation are dependent upon the nature of the host but more importantly that bacterial gene deletion can result in increased host pathology without an increase in bacterial load, crucial considerations for vaccine design.

Inhibitory effects of B cells on antitumor immunity.

B-cell functions in antitumor immunity are not well understood. In this study, we evaluated the role of B cells in the development of antitumor immunity using Friend murine leukemia virus gag-expressing mouse EL-4 (EL-4 gag), D5 mouse melanoma, or MCA304 mouse sarcoma cells. To screen tumors for susceptibility to B-cell-deficient immune environments, spleen cells from naive C57BL/6 [wild-type (WT)] and B-cell knockout (BKO) mice were cultured with irradiated tumor cells in vitro. When cells were stimulated with EL-4 gag or D5 (but not MCA304 tumors), IFN-gamma production from CD8 T cells and natural killer cells was markedly decreased in WT compared with BKO cultures. IFN-gamma production was correlated with CD40 ligand expression on the tumor and inversely with interleukin-10 (IL-10) production by B cells. Sorted WT B cells produced more IL-10 than CD40 knockout (CD40KO) B cells when cocultured with EL-4 gag or D5 (but not MCA304). IFN-gamma production by BKO cells was reduced by the addition of sorted naive WT B cells (partially by CD40KO B cells) or recombinant mouse IL-10. In vivo tumor progression mirrored in vitro studies in that WT mice were unable to control tumor growth whereas EL-4 gag and D5 tumors (but not MCA304) were eliminated in BKO mice. Robust in vivo antitumor CTLs developed only in BKO tumor-challenged mice. Our studies provide the first mechanistic basis for the concept that B-cell depletion could therapeutically enhance antitumor immune responses to certain tumors by decreasing IL-10 production from B cells.

Th1-like cytokine induction by heat-killed Brucella abortus is dependent on triggering of TLR9.

In this report we provide evidence, for the first time, that bacterial DNA in the context of heat-killed Brucella abortus (HKBA) engages TLR9 in dendritic cells (DC), resulting in a Th1-like cytokine response. This is based on the findings that HKBA induction of IL-12p40 is: 1) abolished in DC from TLR9(-/-) mice; 2) blocked by suppressive oligodeoxynucleotides; 3) simulated by bacterial DNA derived from HKBA; and 4) abrogated by DNase or methylation of the DNA from HKBA. Furthermore, the effect of HKBA can be inhibited by chloroquine, indicating that endosomal acidification is required and supporting the notion that DNA from HKBA is interacting with TLR9 at the level of the endosome, as is the case with CpG oligodeoxynucleotides. In addition to DC, HKBA can elicit IL-12p40 secretion from macrophages, in which case the effect is wholly MyD88 dependent but only partially TLR9 dependent. This probably explains why HKBA effects in vivo are only partially reduced in TLR9(-/-), but absent in MyD88(-/-) mice. Because of their intimate interactions with T cells, the DC response is most likely to be critical for linking innate and adaptive immune responses, whereas the macrophage reaction may play a role in enhancing NK cell and bystander immune responses. In addition to IL-12p40, HKBA induces other Th1-like cytokines, namely, IFN-alpha and IFN-gamma, in a TLR9-dependent manner. These cytokines are important in protection against viruses and bacteria, and their induction enhances HKBA as a potential carrier for vaccines.

The cytotoxic T lymphocyte response against a protein antigen does not decrease the antibody response to that antigen although antigen-pulsed B cells can be targets.

The role of activated CD8+ T cells in shaping the dynamics of in vivo antigen presentation and immune responses is a subject receiving more attention. We studied whether cytotoxic T lymphocyte (CTL) would limit antibody responses by targeting antigen-specific B cells. A modified in vivo CTL assay was developed and used herein to demonstrate cytotoxicity in vivo, and to show that antigen-specific B cells that process exogenous antigen and present peptide in association with MHC class I can be the targets of CD8+ T cells. B cells from C57BL/6 mice immunized with ovalbumin (OVA)/alum were pulsed with OVA in vitro, and transferred into C57BL/6 recipient mice that had been immunized with vaccinia virus expressing SIINFEKL minigene to generate CD8+ CTL against K(b)/SIINFEKL. OVA-pulsed B220+ B cells from OVA-immunized mice were killed to a greater extent than B220+ B cells from naïve mice (28+/-20% versus 12+/-16%, p=0.0042). However, mice receiving vaccinia-SIINFEKL and generating CTL, did not appear to target endogenous B cells, since both primary and secondary antibody responses to OVA were unaffected. Our findings indicate that CTL responses to the protein antigen do not interfere with endogenous B cell responses, even though exogenous B cells expressing the CTL epitope can be efficiently lysed.

Programming of CTL with heat-killed Brucella abortus and antigen allows soluble antigen alone to generate effective secondary CTL.

Optimal generation of cytotoxic T cell (CTL) responses continues to be a challenge in the production of vaccines against pathogens such as HIV-1, in part because it is difficult to introduce soluble protein antigens (Ag) into the MHC class I pathway. Using heat-killed Brucella abortus (HKBA) as an adjuvant and ovalbumin (ova) protein as an Ag, we demonstrated that a high dose of Ag was required for systemic and effective CTL. In an adoptive transfer model, primary and secondary ova-specific OT-1 CD8 cell expansion by HKBA plus high dose of ova were partially CD4 T cell-dependent. Interestingly, primary stimulation with HKBA plus ova allowed effective secondary stimulation with ova alone that was equivalent to HKBA plus ova in terms of IFN-gamma production from Ag-specific CD8 cells. Thus a combination of adequate Ag dose, and selection of appropriate adjuvants can meet the threshold not only for primary effective CTL responses to soluble protein Ags but for secondary CTL responses following stimulation with protein Ag alone.

Heat-killed Brucella abortus induces TNF and IL-12p40 by distinct MyD88-dependent pathways: TNF, unlike IL-12p40 secretion, is Toll-like receptor 2 dependent.

Cattle and humans are susceptible to infection with the Gram-negative intracellular bacterium Brucella abortus. Heat-killed B. abortus (HKBA) is a strong Th1 adjuvant and carrier. Previously, we have demonstrated that dendritic cells produce IL-12 in response to HKBA stimulation. In the present study, we use knockout mice and in vitro reconstitution assays to examine the contribution of signaling by Toll-like receptors (TLRs) and their immediate downstream signaling initiator, myeloid differentiation protein MyD88, in the activation following stimulation by HKBA. Our results show that HKBA-mediated induction of IL-12p40 and TNF is dependent on the adapter molecule MyD88. To identify the TLR involved in HKBA recognition, we examined HKBA responses in TLR2- and TLR4-deficient animals. TNF responses to HKBA were TLR4 independent; however, the response in TLR2-deficient mice was significantly delayed and reduced, although not completely abolished. Studies using Chinese hamster ovary/CD14 reporter cell lines stably transfected with either human TLR2 or human TLR4 confirmed the results seen with knockout mice, namely TLR2, but not TLR4, can mediate cellular activation by HKBA. In addition, human embryonic kidney 293 cells, which do not respond to HKBA, were made responsive by transfecting TLR2, but not TLR4 or TLR9. Taken together, our data demonstrate that MyD88-dependent pathways are crucial for activation by HKBA and that TLR2 plays a role in TNF, but not IL-12p40 pathways activated by this microbial product.

Mucosal immunity in mice immunized with HIV-1 peptide conjugated to Brucella abortus.

We have previously shown that a V3-loop peptide from HIV-1 envelope conjugated to heat-inactivated Brucella abortus (Ba) (V3-Ba) is capable of inducing antibodies that neutralize HIV-1 and cytotoxic T cells (CTL) that kill HIV-1-infected targets, even in mice that lack CD4(+) T cells. In this paper we show that intranasal (i.n.) immunization elicits neutralizing antibodies and IFNgamma-secreting T cells at mucosal surfaces. This approach may protect individuals from HIV-1 infection and reduce transmission from infected individuals to their sexual partners and offspring.

Recombinant adeno-associated virus serotype 2 vectors mediate stable interleukin 10 secretion from salivary glands into the bloodstream.

We have constructed a recombinant adeno-associated virus serotype 2 vector encoding human interleukin 10 (rAAVhIL10). IL-10 is a potent antiinflammatory/immune cytokine, which has received growing attention for its therapeutic potential. Human IL-10 (hIL-10) production was virus dose dependent after in vitro infection of HSG cells, a human submandibular gland cell line. The vector-derived hIL-10 produced was biologically active, as the medium from rAAVhIL10-infected HSG cells caused a dose-dependent blockade of IL-12 secretion from spleen cells of IL-10 knockout mice challenged with heat-killed Brucella abortus. Administration of rAAVhIL10 (10(10) genomes per gland) to both mouse submandibular glands led to hIL-10 secretion into the bloodstream (approximately 1-5 pg/ml), that is, in an endocrine manner, which was stable for approximately 2 months. Salivary gland administration of rAAVhIL10 under experimental conditions was more efficacious than intravenous administration (approximately 0.5-0.7 pg/ml). Also, hIL-10 was readily secreted in vitro from organ cultures of minced submandibular glands infected with rAAVhIL10, 6 or 8 weeks earlier. Consistent with these results, hIL-10 mRNA was detected by reverse transcription-polymerase chain reaction in submandibular glands of mice infected with rAAVhIL10 but not from control mice. At these doses, little to no hIL-10 was detected in mouse saliva. Using a rAAV serotype 2 vector encoding beta-galactosidase, we observed that the primary parenchymal target cells were ductal. These findings represent the first report of rAAV use to target exocrine glands for systemic secretion of a therapeutic protein, and support the notion that rAAV serotype 2 vectors may be useful in salivary glands for local (periglandular) and systemic gene-based protein therapeutics.